Image-Based Assay Using Mark-Assisted Machine Learning

ABSTRACT

The present disclosure relates to devices, apparatus and methods of improving the accuracy of an image-based assay. One aspect of the present invention is to sandwich a sample between two plates and add reference marks in the sample areas of the plates, with at least one of the geometric and/optical properties of the reference marks being predetermined and known, and taking images of the sample with the reference marks, and applying a machine learning model in the analysis of the image-based assay.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/483,700, filed on Aug. 5, 2019, which is a § 371 national stageapplication of International Application PCT/US2018/017504 filed on Feb.8, 2018, which claims the benefit of priority to U.S. Provisional PatentApplication No. 62/456,590, filed on Feb. 8, 2017, U.S. ProvisionalPatent Application No. 62/459,554, filed on Feb. 15, 2017, U.S.Provisional Patent Application No. 62/460,075, filed on Feb. 16, 2017,U.S. Provisional Patent Application No. 62/456,504, filed on Feb. 8,2017, U.S. Provisional Patent Application No. 62/460,062, filed on Feb.16, 2017 and U.S. Provisional Patent Application No. 62/457,133, filedon Feb. 9, 2017, the contents of which are relied upon and incorporatedherein by reference in their entirety. The entire disclosure of anypublication or patent document mentioned herein is entirely incorporatedby reference.

FIELD

Among other things, the present invention is related to devices andmethods of performing biological and chemical assays, and computationalimaging.

BACKGROUND

In biological and chemical assays (e.g. diagnostic testing), often itneeds to simple, fast, and sensitive assaying, including imaging. Thepresent invention provides, among other thing, devices and methods forsimple, fast, and sensitive assaying, including imaging.

BRIEF DESCRIPTION OF THE DRAWINGS

The skilled artisan will understand that the drawings, described below,are for illustration purposes only. The drawings are not intended tolimit the scope of the present teachings in any way. The drawings arenot entirely in scale. In the figures that present experimental datapoints, the lines that connect the data points are for guiding a viewingof the data only and have no other means.

FIGS. 1-A, 1-B and 1-C are the schematic illustration of system testingsample in fluorescent illumination mode, according to some embodimentsof the present invention.

FIGS. 2-A, 2-B and 2-C are the schematic illustration of system testingsample in bright-field illumination mode, according to some embodimentsof the present invention.

FIG. 3 is the schematic exploded view of optical adaptor device insystem and system 20, according to some embodiments of the presentinvention.

FIG. 4 is the schematic sectional view showing details of system testingsample in bright-field illumination mode, and particularly of device,according to some embodiments of the present invention.

FIG. 5 is the schematic sectional view showing details of system testingsample in fluorescent illumination mode, and particularly of device,according to some embodiments of the present invention.

FIG. 6-A and FIG. 6-B is the schematic sectional viewing showing thedesign to make lever stop at the pre-defined position when being pulledoutward from the device, according to some embodiments of the presentinvention.

FIG. 7 is the schematic illustration of the structure of the sampleslider holding the QMAX device, according to some embodiments of thepresent invention.

FIG. 8 is the schematic illustration of the moveable arm switchingbetween two pre-defined stop positions, according to some embodiments ofthe present invention.

FIG. 9 is the schematic illustration of how the slider indicates if QMAXdevice is inserted in right direction, according to some embodiments ofthe present invention.

FIGS. 10-A, 10-B and 10-C are the schematic illustration of system forsmartphone colorimetric reader, according to some embodiments of thepresent invention.

FIG. 11 is the schematic exploded view of optical adaptor device insystem, according to some embodiments of the present invention.

FIG. 12 is the schematic sectional view showing details of systemreading a colorimetric card, and particularly of device, according tosome embodiments of the present invention.

FIGS. 13-A, 13-B and 13-C are the schematic illustrations of system forsmartphone colorimetric reader, according to some embodiments of thepresent invention.

FIG. 14 is the schematic exploded view of optical adaptor device insystem, according to some embodiments of the present invention.

FIGS. 15-A, 15-B and 15-C are the schematic views showing details ofsystem reading a colorimetric card, and particularly of device,according to some embodiments of the present invention.

FIG. 16-A shows a tomography device that consists of an imaging sensor,a lens, and a QMAX structure, according to some embodiments of thepresent invention.

FIG. 16-B shows an example of the pillar array pattern of the letter E.

FIG. 16-C shows the thin lens model, which explains the effect of focaldistance on the captured image.

FIG. 16-D shows a captured image of the example pillar array in FIG.16-B by the imaging sensor.

FIG. 16-E shows the diagram of phase image retrieval based scheme.

FIG. 17-A shows analyte detection and localization workflow, whichconsists of two stages, training and prediction, according to someembodiments of the present invention.

FIG. 17-B shows the process to remove one item from an ordered list,according to some embodiments of the present invention.

FIG. 18-A shows an embodiment of a QMAX device used for cell imaging inan open configuration, according to some embodiments of the presentinvention.

FIG. 18-B shows an embodiment of a QMAX device used for cell imaging inan open configuration, according to some embodiments of the presentinvention.

FIG. 18-C shows an embodiment of a QMAX device used for cell imaging ina closed configuration, according to some embodiments of the presentinvention.

FIG. 19-A shows a schematic illustration of the dual camera imagingsystem.

FIG. 19-B shows an example of the dual camera imaging system for largeFOV imaging according to some embodiments of the present invention.

FIG. 19-C shows an example of the dual camera imaging system for dualresolution imaging according to some embodiments of the presentinvention.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

The following detailed description illustrates some embodiments of theinvention by way of example and not by way of limitation. The sectionheadings and any subtitles used herein are for organizational purposesonly and are not to be construed as limiting the subject matterdescribed in any way. The contents under a section heading and/orsubtitle are not limited to the section heading and/or subtitle, butapply to the entire description of the present invention.

The citation of any publication is for its disclosure prior to thefiling date and should not be construed as an admission that the presentclaims are not entitled to antedate such publication by virtue of priorinvention. Further, the dates of publication provided can be differentfrom the actual publication dates which can need to be independentlyconfirmed.

Seven exemplary embodiments are illustrated as followed: one embodimentof optical adaptor for bright-field and fluorescent microscopy imagingattached to a smartphone; one embodiment of optical adaptor forcolorimetric measurement attached to a smartphone using tilted fiber endface as light source; one embodiment of optical adaptor for colorimetricmeasurement attached to a smartphone using side-illumination of aring-shape fiber as light source; one embodiment of device and methodsof tomography; one embodiment of machine learning assisted assay andimaging; one embodiment of device and methods of tissue staining andcell imaging; one embodiment of dual-lens imaging system.

A. Optical Adaptor for Bright-Field and Fluorescent MicroscopeAttachment to Smartphone

Bright-field and fluorescent microscopy are very powerful techniques tolet people examine some property of a sample, which have wideapplications in health monitoring, disease diagnostic, science educationand so on. Conventionally, the taking microscopy images requires,however, expensive microscope and experienced personnel which commonpeople have limited access to. Even though there some recent inventedaccessories which can turn a smartphone into a bright-field microscope,the bright-field microscopy images only give very limited information ofthe sample.

The present invention that is described herein address this problem byproviding a system comprising an optical adaptor and a smartphone. Theoptical adaptor device fits over a smartphone converting it into amicroscope which can take both fluorescent and bright-field images of asample. This system can be operated conveniently and reliably by acommon person at any location. The optical adaptor takes advantage ofthe existing resources of the smartphone, including camera, lightsource, processor and display screen, which provides a low-cost solutionlet the user to do bright-field and fluorescent microscopy.

In this invention, the optical adaptor device comprises a holder framefitting over the upper part of the smartphone and an optical boxattached to the holder having sample receptacle slot and illuminationoptics. In some prior arts (U.S. Pat. No. 2016/029091 and U.S. Pat. No.2011/0292198), their optical adaptor design is a whole piece includingboth the clip-on mechanics parts to fit over the smartphone and thefunctional optics elements. This design has the problem that they needto redesign the whole-piece optical adaptor for each specific model ofsmartphone. But in this present invention, the optical adaptor isseparated into a holder frame only for fitting a smartphone and auniversal optical box containing all the functional parts. For thesmartphones with different dimensions, as long as the relative positionsof the camera and the light source are the same, only the holder frameneed to be redesigned, which will save a lot of cost of design andmanufacture.

The optical box of the optical adaptor comprises: a receptacle slotwhich receives and position the sample in a sample slide in the field ofview and focal range of the smartphone camera; a bright-fieldillumination optics for capturing bright-field microscopy images of asample; a fluorescent illumination optics for capturing fluorescentmicroscopy images of a sample; a lever to switch between bright-fieldillumination optics and fluorescent illumination optics by slidinginward and outward in the optical box.

The receptacle slot has a rubber door attached to it, which can fullycover the slot to prevent the ambient light getting into the optical boxto be collected by the camera. In the prior art (U.S. Pat.2016/0290916), its sample slot is always exposed to the ambient lightwhich won't cause too much problem because it only does bright-fieldmicroscopy. But the present invention can take the advantage of thisrubber door when doing fluorescent microscopy because the ambient lightwould bring a lot of noise to the image sensor of the camera.

To capture good fluorescent microscopy image, it is desirable thatnearly no excitation light goes into the camera and only the fluorescentemitted by the sample is collected by the camera. For all commonsmartphones, however, the optical filter putting in front of the cameracannot block the undesired wavelength range of the light emitted fromthe light source of a smartphone very well due to the large divergenceangle of the beams emitted by the light source and the optical filternot working well for un-collimated beams. Collimation optics can bedesigned to collimated the beam emitted by the smartphone light sourceto address this issue, but this approach increase the size and cost ofthe adaptor. Instead, in this present invention, fluorescentillumination optics enables the excitation light to illuminate thesample partially from the waveguide inside the sample slide andpartially from the backside of the sample side in large obliqueincidence angle so that excitation light will nearly not be collected bythe camera to reduce the noise signal getting into the camera.

The bright-field illumination optics in the adaptor receive and turn thebeam emitted by the light source so as to back-illuminated the sample innormal incidence angle.

Typically, the optical box also comprises a lens mounted in it alignedwith the camera of the smartphone, which magnifies the images capturedby the camera. The images captured by the camera can be furtherprocessed by the processor of smartphone and outputs the analysis resulton the screen of smartphone.

To achieve both bright-field illumination and fluorescent illuminationoptics in a same optical adaptor, in this present invention, a slidablelever is used. The optical elements of the fluorescent illuminationoptics are mounted on the lever and when the lever fully slides into theoptical box, the fluorescent illumination optics elements block theoptical path of bright-field illumination optics and switch theillumination optics to fluorescent illumination optics. And when thelever slides out, the fluorescent illumination optics elements mountedon the lever move out of the optical path and switch the illuminationoptics to bright-field illumination optics. This lever design makes theoptical adaptor work in both bright-field and fluorescent illuminationmodes without the need for designing two different single-mode opticalboxes.

The lever comprises two planes at different planes at different heights.

In some embodiments, two planes can be joined together with a verticalbar and move together in or out of the optical box. In some embodiments,two planes can be separated and each plane can move individually in orout of the optical box.

The upper lever plane comprises at least one optical element which canbe, but not limited to be an optical filter. The upper lever plane movesunder the light source and the preferred distance between the upperlever plane and the light source is in the range of 0 to 5 mm.

Part of the bottom lever plane is not parallel to the image plane. Andthe surface of the non-parallel part of the bottom lever plane hasmirror finish with high reflectivity larger than 95%. The non-parallelpart of the bottom lever plane moves under the light source and deflectsthe light emitted from the light source to back-illuminate the samplearea right under the camera. The preferred tilt angle of thenon-parallel part of the bottom lever plane is in the range of 45 degreeto 65 degree and the tilt angle is defined as the angle between thenon-parallel bottom plane and the vertical plane.

Part of the bottom lever plane is parallel to the image plane and islocated under and 1 mm to 10 mm away from the sample. The surface of theparallel part of the bottom lever plane is highly light absorptive withlight absorption larger than 95%. This absorptive surface is toeliminate the reflective light back-illuminating on the sample in smallincidence angle.

To slide in and out to switch the illumination optics using the lever, astopper design comprising a ball plunger and a groove on the lever isused in order to stop the lever at a pre-defined position when beingpulled outward from the adaptor. This allow the user to use arbitraryforce the pull the lever but make the lever to stop at a fixed positionwhere the optical adaptor's working mode is switched to bright-filedillumination.

A sample slider is mounted inside the receptacle slot to receive theQMAX device and position the sample in the QMAX device in the field ofview and focal range of the smartphone camera.

The sample slider comprises a fixed track frame and a moveable arm:

The frame track is fixedly mounted in the receptacle slot of the opticalbox. And the track frame has a sliding track slot that fits the widthand thickness of the QMAX device so that the QMAX device can slide alongthe track. The width and height of the track slot is carefullyconfigured to make the QMAX device shift less than 0.5 mm in thedirection perpendicular to the sliding direction in the sliding planeand shift less than less than 0.2 mm along the thickness direction ofthe QMAX device.

The frame track has an opened window under the field of view of thecamera of smartphone to allow the light back-illuminate the sample.

A moveable arm is pre-built in the sliding track slot of the track frameand moves together with the QMAX device to guide the movement of QMAXdevice in the track frame.

The moveable arm equipped with a stopping mechanism with two pre-definedstop positions. For one position, the arm will make the QMAX device stopat the position where a fixed sample area on the QMAX device is rightunder the camera of smartphone. For the other position, the arm willmake the QMAX device stop at the position where the sample area on QMAXdevice is out of the field of view of the smartphone and the QMAX devicecan be easily taken out of the track slot.

The moveable arm switches between the two stop positions by a pressingthe QMAX device and the moveable arm together to the end of the trackslot and then releasing.

The moveable arm can indicate if the QMAX device is inserted in correctdirection. The shape of one corner of the QMAX device is configured tobe different from the other three right angle corners. And the shape ofthe moveable arm matches the shape of the corner with the special shapeso that only in correct direction can QMAX device slide to correctposition in the track slot.

FIGS. 1-A, 1-B and 1-C is the schematic illustration of system 19testing sample in fluorescent illumination mode. Particularly, FIGS. 1-Band 1-C are the exploded views of system 19, shown from the front andrear sides respectively. System 19 comprises a smartphone 1; an opticaladaptor device 18 fitting over the upper part of smartphone 1; a sampleslide 5, inserted into receptacle slot 4 of device 18 so that the sampleon sample slide 5 is positioned within the field of view and focal rangeof camera module 1C in smartphone 1. A lever 8 is fully pressed intodevice 18 so that system 19 operates in fluorescent illumination mode. Arubber door 16 attached to device 18 covers receptacle slot 4 aftersample slide 5 is in so as to prevent the ambient light getting intoreceptacle slot 4 to affect the test.

The software (not shown) installed in smartphone 1 analyzes the imagecollected by camera module 1C while light source 1L in smartphone 1 isemitting light, in order to get some property of the sample, and outputsthe results to a display screen 1 f in smartphone 1.

FIGS. 2-A, 2-B and 2-C is the schematic illustration of system 20testing sample in bright-field illumination mode. Particularly, FIGS.2-B and 2-C are the exploded views of system 20, shown from the frontand rear sides respectively. System 20 comprises a smartphone 1; anoptical adaptor device 18 fitting over the upper part of smartphone 1; asample slide 5, inserted into receptacle slot 4 of device 18 so that thesample on sample slide 5 is positioned within the field of view andfocal range of camera module 1C in smartphone 1. A lever 8 is pulledoutward from device 18 and stopped by a stopper (not shown) at apre-designed position in device 18 so that system 20 operates inbright-field illumination mode.

FIG. 3 is the schematic exploded view of optical adaptor device 18 insystem 19 and system 20. Device 18 comprises a holder case 2 fittingover the upper part of smartphone 1; an optical box 3 attached to case 2including a receptacle slot 4, an optics chamber 3C, track 6 b and 6 tallowing lever 8 to slide in, and a rubber door 16 inserted into trench4 s to cover receptacle slot 4. An optics insert 7 is fitted into thetop of optics chamber 3C with an exit aperture 7L and an entranceaperture 7C in it aligning with light source 1L and camera 1C (shown inFIG. 2-B) in smartphone 1. A lens 11 is mounted in entrance aperture 7Cin optics insert 7 and configured so that the sample in sample slide 5inserted into receptacle slot 4 is located within the working distanceof the camera 1C (shown in FIGS. 2-B and 1-B). Lens 11 serves to magnifythe images of the sample captured by camera 1C (shown in FIGS. 2-B and1-B). A long-pass optical filter 12 is mounted on top of lens 11 inentrance aperture 7C. A pair of right angle mirrors 13 and 14 aremounted on the bottom of optics chamber 3C and configured so that mirror13 and mirror 14 are aligned with light source 1L and camera 1C (shownin FIGS. 2-B and 1-B) respectively. Mirror 13 and mirror 14 whoseoperation as bright-field illumination optics in device 18 is describedbelow in FIG. 4. Lever 8 comprise two level bars: the upper-level barcomprises a band-pass optical filter 15 mounted in slot 8 a, and thelower-level bar comprises a light absorber 9 mounted on the horizontalplane 8 b and a reflective mirror 10 mounted on the tilted plane 8c. Theoptical filter 15, light absorber 9 and mirror 10 whose operation asfluorescent illumination optics in device 18 is described below in FIG.5. The upper-level bar of lever 8 slides along track 6 t in box 3 andlower-level bar 8 b and 8 c slides along track 6 b in box 3. Lever 8stops at two different positions in box 3 to switch between bright-fieldillumination optics and fluorescent illumination optics. Lever 8 isfully inserted into box 3 to switch device 18 to work with fluorescentillumination optics. Ball plunger 17 is mounted on the sidewall of track6 t to stop lever 8 at a pre-defined position when lever 8 being pulledoutward from box 3 to switch device 18 to work with bright-fieldillumination optics.

FIG. 4 is the schematic sectional view showing details of system 20testing sample in bright-field illumination mode, and particularly ofdevice 18. This figure illustrates the functionality of the elementsthat were described above with reference to FIG. 3. Lever 8 (shown inFIG. 3) is pulled outward from device 18 and stopped by stopper 17(shown in FIG. 3) at a pre-defined position so that mirror 13 and mirror14 is exposed to and aligned with camera 1C and light source 1L. Lightsource 1L emits light beam BB1 away from smartphone 1. Beam BB1 isdeflected by mirror 14 by 90 degrees to beam BB2 which is furtherdeflected by mirror 13 by 90 degrees to beam BB3. Beam BB3back-illuminates the sample in sample slide 5 in normal incidence angle.Lens 11 creates a magnified images of the sample on the image sensorplane of camera 1C. Smartphone 1 captures and processes the image to getsome property of the sample.

FIG. 5 is the schematic sectional view showing details of system 19testing sample in fluorescent illumination mode, and particularly ofdevice 18. This figure illustrates the functionality of the elementsthat were described above with reference to FIG. 3. Lever 8 (shown inFIG. 3) is fully inserted into device 18 so that light absorber 9 andtilted mirror 10 are under the view of camera 1C and light source 1L,and block the light path between light source 1L and the pair of mirrorsof 13 and 14. And band-pass optical filter 15 is right under the lightsource 1 L. Light source 1L emits light beam BF1 away from smartphone 1.Optical filter 15 allows beam BF1 with specific wavelength range whichmatches the excitation wavelength of the fluorescent sample in sampleslide 5 to go through. Part of beam BF1 illuminates on the edge oftransparent sample slide 5 and couples to waveguide beam BF3 travellingin sample slide 5 and illuminates the sample area under the lens 11.Part of beam BF1 illuminates on mirror 10. Tilted mirror 10 deflectsbeam BF1 to beam BF2 and back-illuminates the sample area in sampleslide 5 right under lens 11 in large oblique angle. The remaining partof beam BF1 with large divergence angle (i.e., beam BF4) illuminates onabsorber 9 and get absorbed so that no reflected light of beam BF4 getsinto the camera 1C in small incidence angle. The light coming from thesample area under the lens 11 goes through the lens 11 and is filteredby long-pass filter 12 so that only light in a specify wavelength rangethat is emitted by the fluorescent sample in sample slide 5 gets intocamera 1C to form an image. Smartphone 1 captures and processes theimage to get some property of the sample. Rubber door 16 is insertedinto device 18 to cover sample slide 5 to prevent ambient light gettinginto device 18 to affect the test.

FIG. 6-A and FIG. 6-B is the schematic sectional viewing showing thedesign to make lever 8 stop at the pre-defined position when beingpulled outward from the device 18. Ball plunger 17 is mounted in thesidewall of track slot 6 t, and a groove 8 g is drilled on the sidewallof lever 8 with the shape matching the shape of the ball in ball plunger17. When lever 8 is being pulled outward from device 18 and has notreach the pre-defined position as shown in FIG. 6-A, the ball in ballplunger 17 in pressed into its body by the sidewall of lever 8 so thatlever 8 can slide along the track 6 t. As shown in FIG. 6-B, when thegroove 8 g on lever 8 reach to the position of ball plunger 17, the ballin ball plunger 17 jump into groove 8 g to stop lever 8.

FIG. 7 is the schematic illustration of the structure of the sampleslider holding the QMAX device. The sample slider comprises a trackframe having a track slot to let QMAX device slide along it, a moveablearm pre-built in the track slot moving together with QMAX device toguide its movement. The moveable arm equipped with a stopping mechanismto make QMAX device stop at two pre-defined stop positions. The widthand height of the track slot is carefully configured to make the QMAXdevice shift less than 0.5 mm in horizontal direction perpendicular tothe sliding direction and shift less than less than 0.2 mm along thethickness direction of the QMAX device.

FIG. 8 is the schematic illustration of the moveable arm switchingbetween two pre-defined stop positions. By pressing the QMAX device andthe moveable arm together to the end of the track slot and thenreleasing, the QMAX card can stop at either position 1 where sample areais out of field of view of smartphone camera for easily taking out theQMAX device from the slider or position 2 where sample area is rightunder the field of view of smartphone camera for capturing image.

FIG. 9 is the schematic illustration of how the slider indicates if QMAXdevice is inserted in right direction. The shape of one corner of theQMAX device is configured to be different from the other three rightangle corners. And the shape of the moveable arm matches the shape ofthe corner with the special shape so that only in correct direction canQMAX device slide to correct position in the track slot. If the QMAXdevice is flipped or inserted from the wrong side, the part of the QMAXdevice outside the slider is longer that when the QMAX device iscorrectly inserted.

When both fluorescent image and bright-field images are available, onecan employ the knowledge of the fluorescent image to process thebright-field image, or employ the knowledge of the bright-field image toprocess the fluorescent image, or collectively process two images. Thefield-of-view of the fluorescent image and bright-field image can bedifferent; thus, the two images are not spatially aligned,pixel-to-pixel.

To solve the mis-alignment between the fluorescent image andbright-field image, one can apply image registration to these twoimages. An image registration finds a geometric transform that relatesthe spatial position from one image to another. Various imageregistration algorithms can be used for aligning a fluorescent image andbright-field image, including but not limited to, feature-point based,cross-correlation based, Fourier alignment based, etc. The imageregistration outputs a geometric transform that maps the spatialposition (coordinate) of one image to another.

After the fluorescent image and bright-field image are aligned, one canutilize the information from two images to refine the processing of oneimage, or process two images collectively.

EXAMPLES

-   A1. An optical adaptor, comprising: a holder frame, and an optical    box removably attached to the holder frame, wherein the holder frame    is configured to removably fit over a mobile device and align the    optical box to a camera and an illumination source integrated in the    mobile device; wherein the optical box comprises sample receptacle    slot and illumination optics.-   B1. An optical system, comprising: the optical adaptor of embodiment    A1; and a QMAX card, which comprises a first plate and a second,    wherein the first plate and the second compresses a liquid sample    into a layer of uniform thickness of less than 200 um; and a slider    that configured to accommodate the QMAX card and to be asserted into    the optical box.-   C1. The adaptor or system of any prior embodiments, wherein the    mobile device is a smart phone.-   C2. The adaptor or system of any prior embodiments, wherein the    holder frame comprises a holder case that is configured to be    replaceable with other holder cases having a different size for    different mobile devices.-   C3. The adaptor or system of any prior embodiments, wherein the    holder frame is sized to removably fit the optical adaptor to an    upper part of the mobile device.-   C4. The adaptor or system of any prior embodiments, wherein the    optical box of the optical adaptor comprises: a receptacle slot that    is configured to receive and position the QMAX card in a sample    slide in the field of view and focal range of the camera; a    bright-field illumination optics that is configured to capture    bright-field microscopy images of the sample; a fluorescent    illumination optics that is configured to capture fluorescent    microscopy images of a sample; and a lever that is configured to    switch between bright-field illumination optics and fluorescent    illumination optics by sliding inward and outward in the optical    box.-   C5. The adaptor or system of any prior embodiments, wherein the    receptor slot comprises a rubber door, which can fully cover the    slot to prevent the ambient light getting into the optical box to be    collected by the camera.-   C6. The adaptor or system of any prior embodiments, wherein the    bright-field illumination optics in the adaptor is configured to    receive and turn the beam emitted by the light source so as to    back-illuminated the sample in normal incidence angle-   C7. The adaptor or system of any prior embodiments, wherein optical    box further comprises a lens mounted in it and aligned with the    camera of the mobile device, which magnifies the images captured by    the camera.-   C8. The adaptor or system of any prior embodiments, wherein the    images captured by the camera are further processed by processors of    mobile device and outputs the analysis result on a screen of mobile    device.-   C9. The adaptor or system of any prior embodiments, wherein the    level is slidable and is configured to achieve both bright-field    illumination and fluorescent illumination optics in the same optical    adaptor.-   C10. The adaptor or system of any prior embodiments, wherein optical    elements of the fluorescent illumination optics are mounted on the    lever and when the lever fully slides into the optical box,-   C11. The adaptor or system of any prior embodiments, wherein the    lever with the fluorescent illumination optics elements block the    optical path of bright-field illumination optics and switch the    illumination optics to fluorescent illumination optics-   C12. The adaptor or system of any prior embodiments, wherein when    the lever slides out, the fluorescent illumination optics elements    mounted on the lever move out of the optical path and switch the    illumination optics to bright-field illumination optics-   C13. The adaptor or system of any prior embodiments, wherein the    lever comprises two planes at different heights.-   C14. The adaptor or system of any prior embodiments, wherein the two    planes are joined together with a vertical bar and move together in    or out of the optical box.-   C15. The adaptor or system of any prior embodiments, wherein the two    planes can be separated and each plane can move individually in or    out of the optical box.-   C16. The adaptor or system of any prior embodiments, wherein the    upper lever plane comprises at least one optical element which can    be, but not limited to be an optical filter.-   C17. The adaptor or system of any prior embodiments, wherein the    upper lever plane moves under the light source and the preferred    distance between the upper lever plane and the light source is in    the range of 0 to 5 mm.-   C18. The adaptor or system of any prior embodiments, wherein part of    the bottom lever plane is not parallel to the image plane.-   C19. The adaptor or system of any prior embodiments, wherein the    surface of the non-parallel part of the bottom lever plane has    mirror finish with high reflectivity larger than 95%.-   C20. The adaptor or system of any prior embodiments, wherein the    non-parallel part of the bottom lever plane moves under the light    source and deflects the light emitted from the light source to    back-illuminate the sample area right under the camera.-   C21. The adaptor or system of any prior embodiments, wherein the    preferred tilt angle of the non-parallel part of the bottom lever    plane is in the range of 45 degree to 65 degree and the tilt angle    is defined as the angle between the non-parallel bottom plane and    the vertical plane.-   C22. The adaptor or system of any prior embodiments, wherein part of    the bottom lever plane is parallel to the image plane and is located    under and 1 mm to 10 mm away from the sample.-   C23. The adaptor or system of any prior embodiments, wherein the    surface of the parallel part of the bottom lever plane is highly    light absorptive with light absorption larger than 95%.-   C24. The adaptor or system of any prior embodiments, wherein the    absorptive surface is to eliminate the reflective light    back-illuminating on the sample in small incidence angle.-   C25. The adaptor or system of any prior embodiments, wherein the    lever comprises a stopper configured to stop the lever.-   C26. The adaptor or system of any prior embodiments, wherein the    stopper comprises a ball plunger and a groove on the lever is used    in order to stop the lever at a pre-defined position when being    pulled outward from the adaptor.-   C27. The adaptor or system of any prior embodiments, wherein the    stopper is configured to allow the user to use arbitrary force the    pull the lever but make the lever to stop at a fixed position where    the optical adaptor's working mode is switched to bright-filed    illumination.-   C28. The adaptor or system of any prior embodiments, wherein the    sample slider is mounted inside the receptacle slot to receive the    QMAX device and position the sample in the QMAX device in the field    of view and focal range of the smartphone camera.-   C29. The adaptor or system of any prior embodiments, wherein the    moveable arm switches between the two stop positions by a pressing    the QMAX device and the moveable arm together to the end of the    track slot and then releasing.-   C30. The adaptor or system of any prior embodiments, wherein he    moveable arm can indicate if the QMAX device is inserted in correct    direction.-   C31. The adaptor or system of any prior embodiments, wherein the    shape of one corner of the QMAX device is configured to be different    from the other three right angle corners.-   C31. The adaptor or system of any prior embodiments, wherein the    shape of the moveable arm matches the shape of the corner with the    special shape so that only in correct direction can QMAX device    slide to correct position in the track slot.-   C32. The adaptor or system of any prior embodiments, wherein the    sample slider comprises a fixed track frame and a moveable arm:-   C33. The adaptor or system of any prior embodiments, wherein he    frame track is fixedly mounted in the receptacle slot of the optical    box; and the track frame has a sliding track slot that fits the    width and thickness of the QMAX device so that the QMAX device can    slide along the track. The width and height of the track slot is    carefully configured to make the QMAX device shift less than 0.5 mm    in the direction perpendicular to the sliding direction in the    sliding plane and shift less than less than 0.2 mm along the    thickness direction of the QMAX device.-   C34. The adaptor or system of any prior embodiments, wherein the    frame track has an opened window under the field of view of the    camera of smartphone to allow the light back-illuminate the sample.-   C35. The adaptor or system of any prior embodiments, wherein the    moveable arm is pre-built in the sliding track slot of the track    frame and moves together with the QMAX device to guide the movement    of QMAX device in the track frame.-   C36. The adaptor or system of any prior embodiments, wherein the    moveable arm equipped with a stopping mechanism with two pre-defined    stop positions.

B. Optical Adaptor for Colorimetric Reader Attachment to Smartphone(Tilted-Fiber-End Illumination)

Colorimetric assay is a very powerful technique having wide applicationsin health monitoring, disease diagnostic, chemical analysis and so on.The key factor to get the accurate colorimetric assay result is toaccurately quantify the color change. Conventionally, the color changeof a colorimetric test strip is analyzed by comparing the color changewith a standard color card. But this comparison is accomplished byhuman's eye and can be easily influenced by the environment lightcondition, which limits the accuracy of quantifying the color change.

The present invention that is described herein address this problem byproviding a system comprising an optical adaptor and a smartphone. Theoptical adaptor device fits over a smartphone converting it into acolorimetric reader which can provide a consistent and uniformillumination to illuminate the front surface of the colorimetric testcard and capture the image of the sample to analyze the color change.This system can be operated conveniently and reliably by a common personat any location. The optical adaptor takes advantage of the existingresources of the smartphone, including camera, light source, processorand display screen, which provides a low-cost solution to accuratelyquantify the color change of a colorimetric assay.

In this invention, the optical adaptor device comprises a holder framefitting over the upper part of the smartphone and an optical boxattached to the holder having sample receptacle slot and illuminationoptics. In some prior arts of attachment adaptor for smartphone, theiradaptor design is a whole piece including both the clip-on mechanicsparts to fit over the smartphone and the functional elements. Thisdesign has the problem that they need to redesign the whole-pieceadaptor for each specific model of smartphone. But in this presentinvention, the optical adaptor is separated into a holder frame only forfitting a smartphone and a universal optical box containing all thefunctional parts. For the smartphones with different dimensions, as longas the relative positions of the camera and the light source are thesame, only the holder frame need to be redesigned, which will save a lotof cost of design and manufacture.

The optical box of the optical adaptor comprises: a receptacle slotwhich receives and position the colorimetric sample in the field of viewand focal range of the smartphone camera; an illumination and imagingoptics to create uniform and consistent illumination on the sampleindependently of any external conditions and capture the sample image.

To capture the sample image to accurately represent the color change, itis desirable that the sample area under the camera is uniformlyilluminated. But for all common smartphones, there is always a distancebetween the light source and the camera. When the sample is placed veryclose to the camera of smartphone, without additional illuminationoptics, the area can be uniformly front-illuminated by the light sourceis right under the light source but not within the field of view of thecamera. To solve this problem, in this present invention, a tiltedlarge-core optical fiber is used to turn the light beam emitted from thelight source to uniformly illuminate the sample area right under thecamera.

And to create a more uniform illumination, it is desirable that thelight beam from an area light source rather than from a LED point lightsource of the smartphone. A separate diffuser placed in front of the endfaces of the optical fiber could be provided for this purpose, but thisapproach increases the elements in the optical adaptor and increase thecost. Instead, in this present invention, both end faces of the opticalfiber are made to have matte finish to serve as the diffuser so that theend face towards the sample can become an area light source to generatemore uniform illumination on the sample.

Typically, the optical box also comprises a lens mounted in it alignedwith the camera of the smartphone, which makes the sample within thefocal range of the camera. The images captured by the camera will befurther processed by the processor of smartphone to analyze the colorchange and outputs the analysis result on the screen of smartphone.

A sample slider is mounted inside the receptacle slot to receive theQMAX device and position the sample in the QMAX device in the field ofview and focal range of the smartphone camera.

The sample slider comprises a fixed track frame and a moveable arm:

The frame track is fixedly mounted in the receptacle slot of the opticalbox. And the track frame has a sliding track slot that fits the widthand thickness of the QMAX device so that the QMAX device can slide alongthe track. The width and height of the track slot is carefullyconfigured to make the QMAX device shift less than 0.5 mm in thedirection perpendicular to the sliding direction in the sliding planeand shift less than less than 0.2 mm along the thickness direction ofthe QMAX device.

The frame track has an opened window under the field of view of thecamera of smartphone to allow the light back-illuminate the sample.

A moveable arm is pre-built in the sliding track slot of the track frameand moves together with the QMAX device to guide the movement of QMAXdevice in the track frame.

The moveable arm equipped with a stopping mechanism with two pre-definedstop positions. For one position, the arm will make the QMAX device stopat the position where a fixed sample area on the QMAX device is rightunder the camera of smartphone. For the other position, the arm willmake the QMAX device stop at the position where the sample area on QMAXdevice is out of the field of view of the smartphone and the QMAX devicecan be easily taken out of the track slot.

The moveable arm switches between the two stop positions by a pressingthe QMAX device and the moveable arm together to the end of the trackslot and then releasing.

The moveable arm can indicate if the QMAX device is inserted in correctdirection. The shape of one corner of the QMAX device is configured tobe different from the other three right angle corners. And the shape ofthe moveable arm matches the shape of the corner with the special shapeso that only in correct direction can QMAX device slide to correctposition in the track slot.

FIGS. 10-A, 10-B and 10-C are the schematic illustration of system 10for smartphone colorimetric reader. Particularly, FIGS. 10-B and 10-Care the exploded views of system 10, shown from the front and rear sidesrespectively. System 10 comprises a smartphone 1; an optical adaptordevice 13 fitting over the upper part of smartphone 1; a colorimetrictest card 137, inserted into receptacle slot 136 of device 13 so thatthe sample area on the sample card 137 is positioned within the field ofview and focal range of camera module 1C in smartphone 1. The software(not shown) installed in smartphone 1 analyzes the image collected bycamera module 1C while light source 1L in smartphone 1 is emittinglight, in order to analyze the color change of the colorimetric test,and outputs the results to a display screen 1 f in smartphone 1.

FIG. 11 is the schematic exploded view of optical adaptor device 13 insystem 10. Device 13 comprises a holder case 131 fitting over the upperpart of smartphone 1; an optical box 132 attached to case 131 includinga receptacle slot 136, an optics chamber 132C. An optics insert 134 isfitted into the top of optics chamber 132C with an exit aperture 134Land an entrance aperture 134C in it aligning with light source 1L andcamera 1C (shown in FIG. 10-B) in smartphone 1. A lens 133 is mounted inentrance aperture 134C in optics insert 134 and configured so that thesample area on colorimetric sample card 137 inserted into receptacleslot 136 is located within the working distance of the camera 1C (shownin FIG. 10-B). A large-core optical fiber 135 is mounted in the exitaperture 134L with tilted angle. Both end faces of fiber 135 are made tohave matte finish. Fiber 135 whose operation as the illumination opticsin device 13 is described below in FIG. B3.

FIG. 12 is the schematic sectional view showing details of system 10reading a colorimetric card, and particularly of device 13. This figureillustrates the functionality of the elements that were described abovewith reference to FIG. 11. Light source 1L emits light beam B1 away fromsmartphone 1. Beam B1 is coupled into the fiber 135 through the firstend face and travels along the direction of fiber 135 and is emitted outfrom the second end face to become beam B2. Beam B2 illuminates thesample area of colorimetric sample card 137 right under the camera 1Cfrom front side to create uniform illumination. Because the end faces offiber 135 are made to be matte and diffusive finish, beam B2 can beregarded as emitting from an area light source, which helps to create amore uniform illumination. The tilt angle in which the fiber 135 ismounted is set to make the central tray of beam B2 illuminates on thearea on the sample card 137 right under the camera. Lens 11 creates animage of the sample area on the image sensor plane of camera 1C.Smartphone 1 captures and processes the image to analyze the colorinformation in the image to quantify the color change of thecolorimetric assay.

C. Optical Adaptor for Colorimetric Reader Attachment to Smartphone(Fiber-Ring Illumination)

Colorimetric assay is a very powerful technique having wide applicationsin health monitoring, disease diagnostic, chemical analysis and so on.The key factor to get the accurate colorimetric assay result is toaccurately quantify the color change. Conventionally, the color changeof a colorimetric test strip is analyzed by comparing the color changewith a standard color card. But this comparison is accomplished byhuman's eye and can be easily influenced by the environment lightcondition, which limits the accuracy of quantifying the color change.

The present invention that is described herein address this problem byproviding a system comprising an optical adaptor and a smartphone. Theoptical adaptor device fits over a smartphone converting it into acolorimetric reader which can provide a consistent and uniformillumination to illuminate the front surface of the colorimetric testcard and capture the image of the sample to analyze the color change.This system can be operated conveniently and reliably by a common personat any location. The optical adaptor takes advantage of the existingresources of the smartphone, including camera, light source, processorand display screen, which provides a low-cost solution to accuratelyquantify the color change of a colorimetric assay.

In this invention, the optical adaptor device comprises a holder framefitting over the upper part of the smartphone and an optical boxattached to the holder having sample receptacle slot and illuminationoptics. In some prior arts of attachment adaptor for smartphone, theiradaptor design is a whole piece including both the clip-on mechanicsparts to fit over the smartphone and the functional elements. Thisdesign has the problem that they need to redesign the whole-pieceadaptor for each specific model of smartphone. But in this presentinvention, the optical adaptor is separated into a holder frame only forfitting a smartphone and a universal optical box containing all thefunctional parts. For the smartphones with different dimensions, as longas the relative positions of the camera and the light source are thesame, only the holder frame need to be redesigned, which will save a lotof cost of design and manufacture.

The optical box of the optical adaptor comprises: a receptacle slotwhich receives and position the colorimetric sample in the field of viewand focal range of the smartphone camera; an illumination and imagingoptics to create uniform and consistent illumination on the sampleindependently of any external conditions and capture the sample image.

To capture the sample image to accurately represent the color change, itis desirable that the sample area under the camera is uniformlyilluminated. But for all common smartphones, the light source is alwaysa point source and mounted next to the camera with some distance, whichmeans the light source is not central symmetric relative to the camera.This causes the problem that, when the sample is placed very close tothe camera of smartphone, without the help of additional illuminationoptics, the illumination pattern on the front surface of a sample in thefield of view of the camera will have a gradient intensity change in alinear direction. Hence, it is desirable to create a light source withlarge emitting area and central symmetric to the camera. To achieve thispurpose, in this present invention, a plastic side-emitting fiber ringis put around the smartphone camera to make the fiber ring centralsymmetric relative to the camera. And the two end faces of the fiberring are mounted towards the light source of the smartphone. This willconvert the original single point light source to infinite number ofsmall light sources having nearly equal luminous intensity distributedon a circle with equal distance from the smartphone camera. The lightemitted from the side wall of the ring fiber further goes through adiffusive film to increase the emitting area and make the illuminationmore even. The sample area right under the camera is uniformlyfront-illuminated by the designed illumination optics based onside-emitting fiber ring.

Because how the color of a colorimetric sample is represented greatlydepends on the illumination condition, it is important to control theillumination in the optical box consistent independently to any externallight conditions. To solve this problem, the receptacle slot has arubber door attached to it, which can fully cover the slot to preventthe environmental light getting into the optical box to result in changeof the illumination condition.

Typically, the optical box also comprises a lens mounted in it alignedwith the camera of the smartphone, which makes the sample within thefocal range of the camera. The images captured by the camera will befurther processed by the processor of smartphone to analyze the colorchange and outputs the analysis result on the screen of smartphone.

A sample slider is mounted inside the receptacle slot to receive theQMAX device and position the sample in the QMAX device in the field ofview and focal range of the smartphone camera.

The sample slider comprises a fixed track frame and a moveable arm:

The frame track is fixedly mounted in the receptacle slot of the opticalbox. And the track frame has a sliding track slot that fits the widthand thickness of the QMAX device so that the QMAX device can slide alongthe track. The width and height of the track slot is carefullyconfigured to make the QMAX device shift less than 0.5 mm in thedirection perpendicular to the sliding direction in the sliding planeand shift less than less than 0.2 mm along the thickness direction ofthe QMAX device.

The frame track has an opened window under the field of view of thecamera of smartphone to allow the light back-illuminate the sample.

A moveable arm is pre-built in the sliding track slot of the track frameand moves together with the QMAX device to guide the movement of QMAXdevice in the track frame.

The moveable arm (also called “lever”) equipped with a stoppingmechanism with two pre-defined stop positions. For one position, the armwill make the QMAX device stop at the position where a fixed sample areaon the QMAX device is right under the camera of smartphone. For theother position, the arm will make the QMAX device stop at the positionwhere the sample area on QMAX device is out of the field of view of thesmartphone and the QMAX device can be easily taken out of the trackslot.

The moveable arm switches between the two stop positions by a pressingthe QMAX device and the moveable arm together to the end of the trackslot and then releasing.

The moveable arm can indicate if the QMAX device is inserted in correctdirection. The shape of one corner of the QMAX device is configured tobe different from the other three right angle corners. And the shape ofthe moveable arm matches the shape of the corner with the special shapeso that only in correct direction can QMAX device slide to correctposition in the track slot.

Some Embodiments 1. Fiber Ring-Illuminator

In some embodiments of optical assembly, wherein: the radius of the sideillunmring fiber is 10 mm; the diameter of ring fiber can be at least 5mm, 10 mm, 15 mm, 20 mm, 25 mm, 30 mm, 40 mm, 50 mm, 60 mm, 80 mm, or100 mm, or in a range between any of the two values; the diameter of thecross-section of the ring fiber can be at least 0.5 mm, 1.0 mm, 1.5 mm,2.0 mm, 2.5 mm, 3 mm, 4 mm, 5 mm, 6 mm, 8 mm, or 10 mm, or in a rangebetween any of the two values.

In some embodiments of optical assembly, wherein the external imagerlens has a diameter of 6 mm; the diameter of the imager lens can be atleast 2 mm, 3 mm, 4 mm, 5 mm, 10 mm, 15 mm, 20 mm, 25 mm, 30 mm, 40 mm,or 50 mm, or in a range between any of the two values.

In some embodiments of optical assembly, wherein the ring fiber can beused in combination with micro-lens array or be replace by a micro-lensarray;

In some embodiments of optical assembly, wherein the optical assemblycomprises a light diffuser plate between the sample and the ring fiber,wherein the light diffusive plate has an aperture configured to alignedwith the camera.

In some embodiments of optical assembly, wherein the length of one sideof the diffusive plate can be at least 5 mm, 10 mm, 15 mm, 20 mm, 25 mm,30 mm, 40 mm, 50 mm, 100 mm, 150 mm, or 200 mm, or in a range betweenany of the two values, wherein the thickness of the diffusive plate canbe at least 2 mm, 3 mm, 4 mm, 5 mm, 10 mm, 15 mm, or 20 mm, or in arange between any of the two values.

In some embodiments of optical assembly, wherein the distance betweenthe diffusive plate and ring fiber can be at least 1 mm, 10 mm, 15 mm,20 mm, 25 mm, 30 mm, 40 mm, 50 mm, 100 mm, or in a range between any ofthe two values.

The optical assembly of claim 2, wherein the distance between the sampleand ring fiber can be at least 2 mm, 10 mm, 15 mm, 20 mm, 25 mm, 30 mm,40 mm, 50 mm, 100 mm, 150 mm, 200 mm, or in a range between any of thetwo values.

Lever:

-   -   1. The optical assembly of claim 3, wherein the distance between        first planar plane on the moveable arm and the light source can        be at least 0.5 mm, 2 mm, 4 mm, 8 mm, 10 mm, 20 mm, 50 mm, 100        mm or in a range between any of the two values.    -   2. The optical assembly of claim 3, wherein the distance between        first planar plane and the second planar plane of the moveable        arm can be at least 5 mm, 10 mm, 15 mm, 20 mm, 40 mm, 100 mm,        200 mm, or in a range between any of the two values.    -   3. The optical assembly of claim 5, wherein the distance that        the moveable arm needs to move to switch between different        positions can be at least 1 mm, 5 mm, 15 mm, 20 mm, 40 mm, 100        mm, or in a range between any of the two values.    -   4. The optical assembly of claim 3, wherein the second planar        plane is connected to a tilted plane, wherein a mirror is        mounted on the tilted plane    -   5. The optical assembly of claim 4, wherein the preferred tilt        angle of the tilted plane can be at least 10 degree, 30 degree,        60 degree, 80 degree, or in a range between any of the two        values, and the tilt angle is defined as the angle between the        second planar plane and tilted plane.

FIGS. 13-A, 13-B and 13-C are the schematic illustrations of system 10for smartphone colorimetric reader. Particularly, FIG. 13-B and FIG.13-C are the exploded views of system 10, shown from the front and rearsides respectively. System 10 comprises a smartphone 1; an opticaladaptor device 13 fitting over the upper part of smartphone 1; acolorimetric sample card 138, inserted into receptacle slot 137 ofdevice 13 so that the sample area on the sample card 138 is positionedwithin the field of view and focal range of camera module 1C insmartphone 1. A rubber door 139 attached to device 18 covers receptacleslot 137 after sample card 138 is in so as to prevent the ambient lightgetting into optical adaptor 13 to affect the test. The software (notshown) installed in smartphone 1 analyzes the image collected by cameramodule 1C while light source 1L in smartphone 1 is emitting light, inorder to analyze the color change of the colorimetric test, and outputsthe results to a display screen 1 f in smartphone 1.

FIG. 14 is the schematic exploded view of optical adaptor device 13 insystem 10. Device 13 comprises a holder case 131 fitting over the upperpart of smartphone 1; an optical box 132 attached to case 131 includinga receptacle slot 137, an optics chamber 132C and a rubber door 139inserted into trench 137s to cover receptacle slot 137. An optics insert134 is fitted into the top of optics chamber 132C with an exit aperture134L and an entrance aperture 134C in it aligning with light source 1 Land camera 1C (shown in FIG. 13-B) in smartphone 1. A lens 133 ismounted in entrance aperture 134C in optics insert 134 and configured sothat the sample area on colorimetric sample card 138 inserted intoreceptacle slot 137 is located within the working distance of the camera1C (shown in FIG. 13-B). A side-emitting optical fiber ring 135 ismounted in optics insert 134 configured to make the camera 1C in thecenter of the fiber ring 135. Both end faces of optical fiber ring 135are mounted in exit aperture 134L facing the light source 1 L. A lightdiffuser film 136 is put under the optical fiber ring 135 and has a holeopened for the aperture of lens. Optical fiber ring 135 whose operationas the illumination optics in device 13 is described below in FIG.15-A-c.

FIGS. 15-A, 15-B and 15-C are the schematic views showing details ofsystem 10 reading a colorimetric card, and particularly of device 13.FIG. 15-A is the sectional view showing details of device 13. And FIG.15-B and FIG. 15-C are the schematic views only showing theconfiguration of the optics elements in device 13. These figuresillustrate the functionality of the elements that were described abovewith reference to FIG. 14. The light emitted from light source 1 L iscoupled into side-emitting optical fiber ring 135 from the two end facesof fiber ring 135 and travels inside along the ring. Beam B1 is emittedout from the side wall of fiber ring and go through the diffuser film136. Beam B1 illuminates the sample area of colorimetric sample card 138right under the camera 1C from front side to create uniformillumination. The illuminated sample area absorbs part of beam B1 andreflects the beam B1 to beam B2. Beam B2 is collected by lens 133 andgets into camera 1C Lens 133 creates an image of the sample area on theimage sensor plane of camera 1C. Smartphone 1 captures and processes theimage to analyze the color information in the image to quantify thecolor change of the colorimetric assay.

D. Device and Systems for Tomography

D-1. Tomography Device with QMAX Structure

A tomography device that reconstructs a sliceable virtualthree-dimensional copy of a biological specimen with the highestresolution of nanoscale is disclosed. The device consists of an imagingsensor, a lens, and a QMAX device, as in FIG. 16-A.

The QMAX device has a periodic pillar array. A biological specimen iscontained in the QMAX device. An index-matching liquid can be used toreduce the scattering of light, and reduce heterogeneities of refractiveindex throughout the specimen. The QMAX structure enhances the detectionsensitivity of six (or more) orders of magnitude.

D-2. Calibration Based on QMAX Structure

The pillar array has a metallic disk on top of each pillar. The metallicdisk provides a calibration signal for both spatial and heightcalibration for images captured by the imaging sensor. The shape of themetallic disk can be designed to facilitate a fast calibration. Forexample, the shape of the metallic disk can be like the letter E; such apillar array is illustrated in FIG. 16-B.

When the imaging sensor capture an image on the QMAX structure, with orwithout a biological specimen, the captured image can be calibratedspatially and the focus distance of the camera can also be quantitivelycalibrated.

For spatial calibration, the captured image goes through an objectdetection. The object detection scheme can be a template matching, anoptical character recognition, a shape detection, or other schemes thatare used in the field. The object detection retrieves the orientation ofthe detected pattern, which in the example of FIG. 16-B is the letter E.With the orientation parameter, spatial calibration is achieved througha two-dimensional geometric transform.

We disclose a quantitative calibration of the focus distance with thepillar array. The effect of focal distance on the captured image can beexplained by the thin lens model, as shown in FIG. 16-C. If the sensingdevice is at a distance δ from the focus plane, point Q will beprojected onto a circle of diameter kσ, and its radiance will spreadover this circle, with Q being defocused. The location v of the focalplane depends on the focal length of the lens, f, and the distance fromthe object, u. The relationship between these three variables is givenby the well-known Gauss lens law or thin lens equation: 1/f=1/u+1/v.

We measure the degree of focus on the captured image, and deduct thefocus plane location. The focus degree measures the focus level eitherthe whole image or every image pixel. A wide variety of algorithms andoperators have been pro-posed in the literature to measure the focusdegree, such as gradient-based, Laplacian-based, wavelet-based,statistics-based, Cosine transform/Fourier transform based, etc.

The focus degree of the pillar array captured at different focus planescan be pre-measured and stored in a look up table. When the imagingsensor captures a new image of the pillar array, for example, FIG. 16-Dshows a captured image of the example pillar array in FIG. 16-B, wecompute the focus degree of the newly captured image, refer the focusdegree to the look up table, and find its corresponding focal planelocation.

D-3. Tomography System

The goal of tomography is to reconstruct a three-dimensional volume of abiological specimen through several projections of it. An end-to-endtomography system includes light source, imaging, and three-dimensionalreconstruction.

Light Source

The light captured by the imaging sensor can be refracted from thespecimen, emitted from the specimen, etc.

Imaging

The imaging part captures projection on the imaging sensor. Theprojections can be captured at different focus distance, differentangles, from different illumination, etc.

Several images can be captured at different focus distances. The lensmoves towards or backward the QMAX structure at a stepsize or a multipleof stepsize. The value of the stepsize and the movement of the lens canbe controlled by hardware or software through an application programinterface. The image sensor records the captured image.

Several images can be captured at different angles. The specimen isrotated and optical images are captured that approximate straight-lineprojections through it. The specimen is rotated to a series of angularpositions, and an image is captured at each orientation. The apparatusis carefully aligned to ensure that the axis of rotation isperpendicular to the optical axis, so that projection data pertaining toeach plane is collected by the imaging sensor. The focal plane can bepositioned halfway between the axis of rotation and the QMAX cardclosest to the lens. This means that every image contains both focuseddata from the front half of the specimen (the half closest to the lens),and out-of-focus data from the back half of the specimen. The focuseddata will be utilized for three-dimensional volume reconstruction, whilethe out-of-focus data will not be used. A band-pass filter can beequipped to select the focused data.

Optical projection tomography is performed using standard tomographicalgorithms. Due to the position of the focal plane relative to the axisof rotation, two images taken 180 degrees apart from each other will befocused on different parts of the specimen. Limiting the back-projectionto the region corresponding to the focused part of the specimen improvesthe quality of the results. As data is accumulated for the variousorientations through the specimen, a semi-disc mask, which acts as aband-pass filter, can be rotated to ensure that only focused data isback-projected.

Several images can be captured at different illumination. Quantitativephase images from time-dependent interference patterns induced by thefrequency shifting of a reference beam relative to the sample beam canbe obtained. A galvanometer-mounted tilting mirror can be used to varythe angle of illumination. A laser beam passes through two acousto-opticmodulators which shift the frequency of the laser beam. A second beamsplitter recombines the specimen and reference laser beams, forming aninterference pattern which is captured at the imaging sensor. Phaseimages are then calculated by applying phase-shifting interferometry.For near-plane wave illumination of a thin specimen with small indexcontrast, the phase of the transmitted field is to a good approximationequal to the line integral of the refractive index along the path ofbeam propagation. Therefore, the phase image can simply be interpretedas the projection of refractive index.

Besides a band-pass filter, various imaging filters can be used duringimage captures, for the purpose of (including but not limited to):

-   -   (1) signal selection, thereby portion of the captured image is        selected;    -   (2) signal enhancement, thereby portion or whole of the captured        image is enhanced;    -   (3) signal transformation, thereby portion or whole of the        captured image is transformed into another representation, such        as frequency representation, multi-scale representation, etc.;    -   (4) signal replication, thereby portion of the captured image is        replaced by another portion of the captured image, or by the        representation of another portion of the captured image;    -   (5) or any combination of (1)-(4).

Captured images can be enhanced through filtering, such as contrastenhancement, color enhancement, noise reduction, etc. It can increasethe dynamic range of pixel intensities, adjust color temperature, boostthe signal to noise ratio, etc.

Captured images can be transformed into another representation, whichcan be more suitable for the three-dimension reconstruction. It can betransformed into a different format (8 bit to 16 bit, integer tofloating point, etc.), different color space (RGB to HSV, etc.),different domain (spatial domain to frequency domain, etc.), etc.

Portion of captured images can be replaced by another portion (ortransformation of another portion) of captured images. It can be aspatial region, which is replaced by the transformation of anotherregion, such as a reflective extension around the boundary, etc. It canbe a frequency subband, which is replaced by the transformation ofanother frequency subband, such as the high frequency subband isreplaced by an estimation from the low frequency subband, etc.

Three-Dimensional Reconstruction

Reconstructing a three-dimensional volume of the biological specimenfrom its projection is an inverse problem. The three-dimensional volumereconstruction can employ a phase image retrieval scheme, aback-projection scheme, non-linear approximation scheme, optimizationscheme, etc.

When several images are captured at different focus distances, wecompute the focus degrees of these images, and list these focus degreesas a vector. Then we refer the vector with the look up table, and findtheir corresponding focal plane distances. The corresponding can bedistance based, correlation based, or other criteria to select the bestmatch.

A diagram of phase image retrieval based scheme is shown in FIG. 16-E.It consists of four components:

-   -   Focus distance calculation    -   Phase image retrieval    -   Height estimation    -   Three-dimensional volume reconstruction

The second component, phase retrieval is through a quantitative phaseimaging technique, based on the transport of intensity (TIE) equation.The TIE equation states

${k\frac{\partial{I\left( {x,y} \right)}}{\partial z}} = {{- \nabla} \cdot \left\lbrack {{I\left( {x,y} \right)}{\nabla{\varphi\left( {x,y} \right)}}} \right\rbrack}$

where

$\frac{\partial I}{\partial z}$

indicates the intensity gradient which can be computed from themulti-focal images, k is the wave number and φ is the sample phasedistribution.

The TIE equation could be solver using fast Fourier transform, discretecosine transform; see for example, “Boundary-artifact-free phaseretrieval with the transport of intensity equation: fast solution withuse of discrete cosine transform”, C. Zuo, Q. Chen, and A. Asundi,Optics Express, Vol. 22, No. 8, April 2014. The phase image φ isretrieved from the TIE equation.

Given the phase image, we estimate the height (thickness) of thebiological specimen. Recall that for a sample with a thickness of t anda refractive index of n, the corresponding optical path length Lp is

L _(p) =t×n

The height of the biological specimen can be computed, with a knownrefractive index.

Going further, the three-dimensional volume of the biological specimencan be reconstructed.

The back-projection algorithm is commonly used in three-dimensionalreconstruction in tomography. It includes Fourier transform basealgorithm, filtered back projection algorithm, back projection andfiltering algorithm, and iterative algorithm.

When the position of the focal plane relative to the axis of rotationdiffers, two images taken 180 degrees apart from each other will befocused on different parts of the specimen. To compensate, a half-planeadjusted back projection algorithm can be employed. Thus, limiting theback-projection to the region corresponding to the focused part of thespecimen will improve the quality of the results. As data is accumulatedfor the various orientations through the specimen, a semi-disc mask canbe rotated to ensure that only focused data is back-projected.

As another embodiment of the back-projection algorithm, a procedurebased on the filtered back-projection method can be applied. A discreteinverse Radon transform is applied to every x−θ slice in the beamrotation direction, with x, the coordinate in the tilt direction and θ,the relative angle of laser beam direction to the optic axis of theobjective lens. To compensate for the angle between imaging andillumination directions, the x values is divided by cos θ. To reduce theeffects of the missing projections, an iterative constraint method canbe applied.

For the inverse problem to reconstruct a three-dimensional volume fromits projection, the resulting three-dimensional volume can be blurred. Aramp filter can be used to remove or reduce the blurriness.

Beside the deblurring filter, various imaging filters can be used forthree-dimensional volume reconstruction, for (including but not limitedto):

-   -   (1) signal selection, where portion of the image or image volume        is selected;    -   (2) signal enhancement, where portion or whole of the image or        image volume is enhanced;    -   (3) signal transformation, where portion or whole of the image        or image volume is transformed into another representation, such        as frequency representation, multi-scale representation, etc.;    -   (4) signal replication, where portion of the image or image        volume is replaced by another portion of the captured image, or        by the representation of another portion of the captured image;    -   (5) or any combination of (1)-(4)

D-4. Examples of Present Invention

-   DA1. A device for sample imaging, comprising a QMAX device and an    imager, wherein: the QMAX device comprises: a first plate, a second    plate, and spacers, wherein: the plates are movable relative to each    other into different configurations; one or both plates are    flexible; each of the plates has, on its respective inner surface, a    sample contact area for contacting a deformable sample; one or both    of the plates comprise the spacers that are fixed with a respective    plate; the spacers have a predetermined substantially uniform height    and a predetermined inter-spacer-distance; and at least one of the    spacers is inside the sample contact area; wherein one of the    configurations is an open configuration, in which: the two plates    are separated apart, the spacing between the plates is not regulated    by the spacers, and the sample is deposited on one or both of the    plates; and wherein another of the configurations is a closed    configuration which is configured after the sample deposition in the    open configuration; and in the closed configuration: at least part    of the sample is compressed by the two plates into a layer of    uniform thickness, wherein the uniform thickness of the layer is    confined by the inner surfaces of the two plates and is regulated by    the plates and the spacers; and the imager is configured to capture    an image of signals emanating from at least part of the layer of    uniform thickness.-   DB1. A system for tomography, comprising a QMAX device, an imager, a    holder, and a control device, wherein: the QMAX device comprises: a    first plate, a second plate, and spacers, wherein: the plates are    movable relative to each other into different configurations; one or    both plates are flexible; each of the plates has, on its respective    inner surface, a sample contact area for contacting a deformable    sample; one or both of the plates comprise the spacers that are    fixed with a respective plate; the spacers have a predetermined    substantially uniform height and a predetermined    inter-spacer-distance; and at least one of the spacers is inside the    sample contact area; wherein one of the configurations is an open    configuration, in which: the two plates are separated apart, the    spacing between the plates is not regulated by the spacers, and the    sample is deposited on one or both of the plates; and wherein    another of the configurations is a closed configuration which is    configured after the sample deposition in the open configuration;    and in the closed configuration: at least part of the sample is    compressed by the two plates into a layer of uniform thickness,    wherein the uniform thickness of the layer is confined by the inner    surfaces of the two plates and is regulated by the plates and the    spacers; the imager comprises an image sensor and a lens, wherein:    the lens is configured to focus signals emanating from at least part    of the layer of uniform thickness and project the focused signals to    the image sensor, and the image sensor is configured to capture    images of said focused signals; the holder is configured to adjust    relative position between the QMAX device and the imager; and the    control device comprises hardware and software for controlling    and/or deducing the position adjustment made by the holder, and    receiving and reconstructing said images into a three-dimensional    volume.-   DBB1. A system for tomography, comprising a QMAX device, an imager,    a holder, and a control device, wherein: the QMAX device comprises:    a first plate, a second plate, and spacers, wherein: the plates are    movable relative to each other into different configurations; one or    both plates are flexible; each of the plates has, on its respective    inner surface, a sample contact area for contacting a deformable    sample; one or both of the plates comprise the spacers that are    fixed with a respective plate; the spacers have a predetermined    substantially uniform height and a predetermined    inter-spacer-distance; and at least one of the spacers is inside the    sample contact area; wherein one of the configurations is an open    configuration, in which: the two plates are separated apart, the    spacing between the plates is not regulated by the spacers, and the    sample is deposited on one or both of the plates; and wherein    another of the configurations is a closed configuration which is    configured after the sample deposition in the open configuration;    and in the closed configuration: at least part of the sample is    compressed by the two plates into a layer of uniform thickness,    wherein the uniform thickness of the layer is confined by the inner    surfaces of the two plates and is regulated by the plates and the    spacers; the imager is capable of changing the focal plane and    comprises an image sensor and a lens, wherein: the lens is    configured to focus signals emanating from at least part of the    layer of uniform thickness and project the focused signals to the    image sensor, and the image sensor is configured to capture images    of said focused signals; the lens is a single lens or a compound    lens consisting several lenses; at least one element lens in the    lens is moveable to change the distance from the image sensor to    change the focal plane of the imager; and the moveable lens can be    driven by stepper motor and/or electromagnetic force, which is    computerized or manually controlled. and the control device    comprises hardware and software for controlling and/or deducing the    position adjustment made by the holder, and receiving and    reconstructing said images into a three-dimensional volume.-   DC1. A method of tomography, comprising the steps of: depositing a    sample onto the QMAX device of any prior device or system    embodiment;    -   (a) after (a), using the two plates of the QMAX device to        compress at least part of the sample into a layer of        substantially uniform thickness that is confined by the sample        contact surfaces of the plates, wherein the uniform thickness of        the layer is regulated by the spacers and the plates, wherein        the compressing comprises:        -   bringing the two plates together; and        -   conformable pressing, either in parallel or sequentially, an            area of at least one of the plates to press the plates            together to a closed configuration, wherein the conformable            pressing generates a substantially uniform pressure on the            plates over the at least part of the sample, and the            pressing spreads the at least part of the sample laterally            between the sample contact surfaces of the plates, and            wherein the closed configuration is a configuration in which            the spacing between the plates in the layer of uniform            thickness region is regulated by the spacers;    -   (b) capturing an image, using the imager of any prior device or        system embodiment, of signals emanating from at least part of        the layer of uniform thickness;    -   (c) adjusting relative position between the QMAX device and        imager, repeating step (c); and    -   (d) after a series of steps (c), reconstructing the captured        images into a three-dimensional volume of said at least part of        the layer,

wherein a conformable pressing is a method that makes the pressureapplied over an area is substantially constant regardless the shapevariation of the outer surfaces of the plates; and

wherein the parallel pressing applies the pressures on the intended areaat the same time, and a sequential pressing applies the pressure on apart of the intended area and gradually move to other area.

-   DCC1. A method to take images at different focal planes, comprising    steps of:    -   (a) computerized or manually controlling the moveable lens in        the imager to the initial position;    -   (b) corresponding the moveable lens position to the position of        the focal plane;    -   (c) capturing the image using the image sensor in the imager and        record the position of focal plane;    -   (d) computerized or manually adding a step displacement to move        the moveable lens to the next position    -   (e) repeat step (b) to (d)    -   (f) after a series of step (e), several images at different        focal planes are captured.-   DA21. The device of any prior embodiment, wherein QMAX device    further comprises a dry reagent coated on one or both plates that    stains the sample.-   DA22. The device of any prior embodiment, wherein:

i. one or both plate sample contact areas comprise one or a plurality ofbinding sites that each binds and immobilizes a respective analyte; or

ii. one or both plate sample contact areas comprise, one or a pluralityof storage sites that each stores a reagent or reagents; wherein thereagent(s) dissolve and diffuse in the sample during or after step (c),and wherein the sample contains one or plurality of analytes; or

iii. one or a plurality of amplification sites that are each capable ofamplifying a signal from the analyte or a label of the analyte when theanalyte or label is 500 nm from the amplification site; or

iv. any combination of i to iii.

-   DA23. The device of any prior embodiment, wherein the imager further    comprises a light source that provides light for illumination or    excitation of the layer of uniform thickness for the imaging.-   DA24. The device of embodiment DA23, wherein the light source is    selected from the group consisting of: LED, laser, incandescent    light, and any combination thereof.-   DB2. The system of embodiment DB1, wherein the signals comprise    optical signals selected from the group consisting of: light    reflection, light refraction, light transmission, luminescence    signals, and any combination thereof.-   DB3. The system of any prior embodiment, wherein the imager further    comprises a light source providing light illuminating said layer of    uniform thickness for the imaging, wherein the light source is    selected from the group consisting of: incandescent light, LED, CFL,    laser, and any combination thereof.-   DB4. The system of any prior embodiment, wherein the imager further    comprises a light source providing excitation light that excites    fluorescence emission from said layer of uniform thickness for the    imaging, wherein the light source is a LED and/or a laser.-   DB5. The system of any prior embodiment, wherein the holder is    capable of adjusting the relative position of the lens to the QMAX    device along its optical axis to change focal plane position of the    lens.-   DB6. The system of any prior embodiment, wherein the holder is    capable of adjusting the relative position between the lens and the    QMAX device to change imaging angle, wherein the imaging angle is an    angle between focal plane of the lens and the layer of uniform    thickness.-   DB7. The system of any prior embodiment, wherein the imager further    comprises a light source providing illumination light for the    imaging, and wherein the holder is capable of adjusting the relative    position between the light source and the QMAX device to change    angle of incidence of the illumination light, wherein the angle of    incidence is the angle between the illumination light and a line    perpendicular to the layer of uniform thickness.-   DB8. The system of any prior embodiment, wherein the control device    comprises hardware and software for sending a command that defines    the position adjustment to the holder, and wherein the holder is    configured to receive said command and make the adjustment with no    more than 10% deviation.-   DB9. The system of any prior embodiment, wherein the control device    comprises hardware and software for sending a command that defines    the position adjustment to the holder, and wherein the holder is    configured to receive said command and make the adjustment with no    more than 1% deviation.-   DB10. The system of any of embodiments DB8-DB9, wherein the control    device comprises hardware and software for receiving an input that    defines the position adjustment and converting the input into the    command for the holder to make the adjustment.-   DB11. The system of any prior embodiment, wherein the system further    comprises a plurality of calibration pillars, and wherein:

(1) said plurality of calibration pillars are placed between the samplecontact areas of the two plates at the closed configuration, and havedifferent heights from one another, which are all smaller than theuniform height of the spacers;

(2) said images are captured at different focal planes along a commonoptical axis; and

(3) the control device comprises hardware and software for: (a)calculating a focus score for each of said images; and (b) deducing afocus plane position at which each of said images is captured bycomparing said focus score with a look-up table, wherein the focus scoreis a matrix of focus degrees calculated for each pixel of a capturedimage, wherein the look-up table is predetermined and comprises a row ofpre-determined focus plane positions along said common optical axis anda corresponding row of calibration focus scores, each of the calibrationfocus scores is calculated based on an image of the calibration pillarscaptured at the corresponding pre-determined focus plane.

-   DB12. The system of any prior embodiment, wherein said images are    captured at different focal planes along a common optical axis, and    wherein the control device comprises hardware and software for: (a)    generating a phase image for a biological entity in said at least    part of the layer, wherein the phase image is a phase distribution    calculated based on wavelength of an illuminating light used for    imaging, at least part of said images that contain signals from the    biological entity, and the focus plane positions at which said    images are respectively captured; and (b) estimating a thickness of    the biological entity based on the phase image and a refractive    index of the sample, wherein the biological entity is a part or    entirety of said at least part of the layer.-   DB13. The system of embodiment DB8, wherein the control device    comprises hardware and software for reconstructing said at least    part of the images into a three-dimensional volume of the biological    entity based on the estimated thickness.-   DB14. The system of any prior embodiment, wherein said images are    captured at different imaging angles, wherein the control device    comprises hardware and software for: (1) knowing or deducing the    imaging angle for each of said images; and (2) reconstructing said    images into a three-dimensional volume based on the known/deduced    imaging angels using a back-projection algorithm, and wherein the    imaging angle is an angle between focal plane of the lens and the    layer of uniform thickness.-   DB15. The system of any prior embodiment, wherein said images are    captured at different angels of incidence of illumination light,    wherein the control device comprises hardware and software for: (1)    knowing or deducing the angle of incidence for each of said images;    and (2) reconstructing said images into a three-dimensional volume    based on the known/deduced angle of incidence using a    back-projection algorithm, and wherein the angle of incidence of the    illumination light is the angle between the illumination light and a    line perpendicular to the layer of uniform thickness.-   DB16. The system of any or embodiments DB14-DB15, wherein the    back-projection algorithm is selected from the group consisting of:    Fourier transform base algorithm, filtered back-projection    algorithm, back-projection and filtering algorithm, iterative    algorithm, and any combination thereof.-   DB17. The system of any prior embodiment, wherein said imager is    equipped with imaging filters, and wherein the captured images are    filtered by the imaging filters and/or software of the control    device for: (1) signal selection, thereby portion of the captured    image is selected; (2) signal enhancement, thereby portion or whole    of the captured image is enhanced; (3) signal transformation,    thereby portion or whole of the captured image is transformed into    another representation, such as frequency representation,    multi-scale representation, etc.; (4) signal replication, thereby    portion of the captured image is replaced by another portion of the    captured image, or by the representation of another portion of the    captured image; or any combination of (1)-(4).-   DB18. The system of any prior embodiment, wherein the control device    further comprises hardware and software for reconstructing said at    least part of the images into a three-dimensional volume, wherein    during three-dimensional volume reconstruction, the images and the    three-dimensional volume are filtered by software for: (1) signal    selection, where portion of the image or image volume is    selected; (2) signal enhancement, where portion or whole of the    image or image volume is enhanced; (3) signal transformation, where    portion or whole of the image or image volume is transformed into    another representation, such as frequency representation,    multi-scale representation, etc.; (4) signal replication, where    portion of the image or image volume is replaced by another portion    of the captured image, or by the representation of another portion    of the captured image; or any combination of (1)-(4)-   DC2. The method of embodiment DC1, further comprising: before step    (c), staining the sample with a dye.-   DC3. The method of any prior method embodiment, wherein during the    step (b), the conformable pressing is by human hand.-   DC4. The method of any prior method embodiment, wherein the    conformable pressing of step (d) is provided by a pressured liquid,    a pressed gas, or a conformal material.-   DC5. The method of any prior method embodiment, wherein the    adjusting step (d) comprises adjusting the relative position of the    lens to the QMAX device along its optical axis to change focal plane    position of the lens.-   DC6. The method of any prior method embodiment, wherein the    adjusting step (d) comprises adjusting the relative position between    the lens and the QMAX device to change imaging angle, wherein the    imaging angle is an angle between focal plane and the layer of    uniform thickness.-   DC7. The method of any prior method embodiment, wherein the imager    further comprises a light source providing illumination light for    the imaging, and wherein the adjusting step (d) comprises adjusting    the relative position of the light source to the QMAX device to    change angle of incidence of the illumination light, wherein the    angle of incidence is the angle between the illumination light and a    line perpendicular to the layer of uniform thickness.-   DC8. The method of any prior method embodiment, wherein the    adjusting step (d) is performed manually.-   DC9. The method of any prior method embodiment, wherein the    adjusting step (d) is performed through a control device operably    coupled to a holder, wherein the control device comprises hardware    and software for receiving an input that defines the position    adjustment and sending a command to the holder, and wherein the    holder is configured to receive said command and make the adjustment    with a deviation no more than 10%.-   DC10. The method of any prior method embodiment, wherein the    adjusting step (d) is performed through a control device operably    coupled to a holder, wherein the control device comprises hardware    and software for receiving an input that defines the position    adjustment and sending a command to the holder, and wherein the    holder is configured to receive said command and make the adjustment    with a deviation no more than 1%.-   DC11. The method of any prior method embodiment, wherein said images    are captured at different focal planes along a common optical axis,    and wherein the reconstructing step (e) comprises: (i) calculating a    focus score for each of said images; and (ii) deducing a focal plane    position at which each of said images is captured by comparing said    focus score with a look-up table, wherein the focus score is a    matrix of focus degrees calculated for each pixel of a captured    image, wherein the look-up table is predetermined and comprises a    row of pre-determined focal plane positions along said common    optical axis and a corresponding row of calibration focus scores,    each of the calibration focus scores is calculated based on an image    of the calibration pillars captured at the corresponding    pre-determined focal plane.-   DC12. The method of any prior method embodiment, wherein said images    are captured at different focal planes along a common optical axis,    and wherein the reconstructing step (e) comprises: (i) generating a    phase image for a biological entity in said at least part of the    layer, wherein the phase image is a phase distribution calculated    based on the wavelength of an illuminating light used for imaging,    at least part of said images that contain signals from the    biological entity, and the focal plane positions at which said    images are captured; and (ii) estimating a thickness of the    biological entity based on the phase image and a refractive index of    the sample, wherein the biological entity is a part or entirety of    said at least part of the layer.-   DC13. The method of embodiment DC12, wherein the reconstructing    step (e) further comprises reconstructing said at least part of the    images into a three-dimensional volume of the biological entity    based on the estimated thickness.-   DC14. The method of any prior method embodiment, wherein said images    are captured at different imaging angles, wherein the reconstructing    step (e) comprises: (i) knowing or deducing the imaging angle for    each of said images; and (ii) reconstructing said images into a    three-dimensional volume based on the known/deduced imaging angels    using a back-projection algorithm, and wherein the imaging angle is    an angle between focal plane of the lens and the layer of uniform    thickness.-   DC15. The method of any prior method embodiment, wherein said images    are captured at different angels of incidence of illumination light,    wherein the reconstructing step (e) comprises: (i) knowing or    deducing the angle of incidence for each of said images; and (ii)    reconstructing said images into a three-dimensional volume based on    the known/deduced angle of incidence using a back-projection    algorithm, and wherein the angle of incidence of the illumination    light is the angle between the illumination light and a line    perpendicular to the layer of uniform thickness.-   DC16. The method of any of embodiments DC14-DC15, wherein the    back-projection algorithm is selected from the group consisting of:    Fourier transform base algorithm, filtered back-projection    algorithm, back-projection and filtering algorithm, iterative    algorithm, and any combination thereof.-   DC17. The method of any prior method embodiment, wherein the sample    is a biological sample selected from the group consisting of: cells,    tissues, bodily fluids, stool, and any combination thereof.-   DC18. The method of any prior method embodiment, wherein the sample    is an environmental sample from an environmental source selected    from the group consisting of a river, lake, pond, ocean, glaciers,    icebergs, rain, snow, sewage, reservoirs, tap water, drinking water,    etc.; solid samples from soil, compost, sand, rocks, concrete, wood,    brick, sewage, the air, underwater heat vents, industrial exhaust,    vehicular exhaust and any combination thereof.-   DC19. The method of any prior method embodiment, wherein the sample    is a foodstuff sample selected from the group consisting of: raw    ingredients, cooked food, plant and animal sources of food,    preprocessed food, partially or fully processed food, and any    combination thereof.-   DC20. The method of any prior method embodiment, wherein the sample    is blood, and the biological entity is red blood cells, white blood    cells, and/or platelets.-   DC21. The method of embodiment DC20, further comprising: calculating    volume of red blood cells, white blood cells, and/or platelets,    based on their respective reconstructed three-dimensional volumes.-   DC22. The method of embodiment DC21, further comprising: based on    the calculated volume, determining a blood test readout selected    from the group consisting of: mean corpuscular volume (MCV),    hematocrit, Red cell distribution width (RDVV), mean platelet volume    (MPV), platelet distribution width (PDVV), immature platelet    fraction (IPF), and any combination thereof.

E. Assay and Imaging Assisted by Machine Learning E-1. QMAX Device forAssay and Imaging

A device for biological analyte detection and localization, comprising aQMAX device, an imager, and a computing unit, is disclosed. A biologicalsample is suspected on the QMAX device. The count and location of ananalyte contained in the sample are obtain by the disclosure.

The imager captures an image of the biological sample. The image issubmitted to a computing unit. The computing unit can be physicallydirectly connected to the imager, connected through network, orin-directly through image transfer.

E-2. Workflow

The disclosed analyte detection and localization employ machine learningdeep learning. A machine learning algorithm is an algorithm that is ableto learn from data. A more rigorous definition of machine learning is “Acomputer program is said to learn from experience E with respect to someclass of tasks T and performance measure P, if its performance at tasksin T, as measured by P, improves with experience E.” It explores thestudy and construction of algorithms that can learn from and makepredictions on data—such algorithms overcome following strictly staticprogram instructions by making data driven predictions or decisions,through building a model from sample inputs.

Deep learning is a specific kind of machine learning based on a set ofalgorithms that attempt to model high level abstractions in data. In asimple case, there might be two sets of neurons: ones that receive aninput signal and ones that send an output signal. When the input layerreceives an input, it passes on a modified version of the input to thenext layer. In a deep network, there are many layers between the inputand output (and the layers are not made of neurons but it can help tothink of it that way), allowing the algorithm to use multiple processinglayers, composed of multiple linear and non-linear transformations.

The disclosed analyte detection and localization workflow consists oftwo stages, training and prediction, as in FIG. 17-A. We describetraining and prediction stages in the following paragraphs.

Training

In the training stage, training data with annotation is fed into aconvolutional neural network. Convolutional neural network a specializedkind of neural network for processing data that has a known, grid-liketopology. Examples include time-series data, which can be thought of asa 1D grid taking samples at regular time intervals, and image data,which can be thought of as a 2D grid of pixels. Convolutional networkshave been tremendously successful in practical applications. The name“convolutional neural network” indicates that the network employs amathematical operation called convolution. Convolution is a specializedkind of linear operation. Convolutional networks are simply neuralnetworks that use convolution in place of general matrix multiplicationin at least one of their layers.

Training data are annotated for the analyte to be detect. Annotationindicates whether or not an analyte presents in a training data.Annotation can be done in the form of bounding boxes which fullycontains the analyte, or center locations of analytes. In the lattercase, center locations are further converted into circles coveringanalytes.

When the size of training data is large, it presents two challenges:annotation (which is usually done by person) is time consuming, and thetraining is computing expensive. To overcome these challenges, one canpartition the training data into patches of small size, then annotateand train on these patches, or a portion of these patches.

Annotated training data is fed into a convolutional neural network formodel training. The output is a model that can be used to makepixel-level prediction on an image. We use the Caffe library with fullyconvolutional network (FCN). Other convolutional neural networkarchitecture can also be used, such as TensorFlow.

The training stage generates a model that will be used in the predictionstage. The model can be repeatedly used in the prediction stage forinput images. Thus, the computing unit only needs access to thegenerated model. It does not need access to the training data, nor thetraining stage has to be run on the computing unit.

Prediction

In the predication stage, a detection component is applied to the inputimage, which is followed by a localization component. The output of theprediction stage is the count of analytes contained in the sample, alongwith the location of each analyte.

In the detection component, an input image, along with the modelgenerated from the training stage, is fed into a convolutional neuralnetwork. The output of the detection stage is a pixel-level prediction,in the form of a heatmap. The heatmap can have the same size as theinput image, or it can be a scaled down version of the input image. Eachpixel in the heatmap has a value from 0 to 1, which can be considered asthe probability (belief) whether a pixel belongs to an analyte. Thehigher the value, the bigger the chance it belongs to an analyte.

The heatmap is the input of the localization component. We disclose analgorithm to localize the analyte center. The main idea is toiteratively detect local peaks from the heatmap. After we find the peak,we calculate the local area surrounding the peak but with smaller value.We remove this region from the heatmap and find the next peak from theremaining pixels. The process is repeated only all pixels are removedfrom the heatmap.

One embodiment of the localization algorithm is to sort the heatmapvalues into a one-dimensional ordered list, from the highest value tothe lowest value. Then pick the pixel with the highest value, remove thepixel from the list, along with its neighbors. Iterate the process topick the pixel with the highest value in the list, until all pixels areremoved from the list.

Algorithm GlobalSearch (heatmap) Input: heatmap Output: loci loci ←{}sort(heatmap) while (heatmap is not empty) { s ← pop(heatmap) D ← {diskcenter as s with radius R} heatmap = heatmap \ D // remove D from theheatmap add s to loci }

After sorting, heatmap is a one-dimensional ordered list, where theheatmap value is ordered from the highest to the lowest. Each heatmapvalue is associated with its corresponding pixel coordinates. The firstitem in the heatmap is the one with the highest value, which is theoutput of the pop(heatmap) function. One disk is created, where thecenter is the pixel coordinate of the one with highest heatmap value.Then all heatmap values whose pixel coordinates resides inside the diskis removed from the heatmap. The algorithm repeatedly pops up thehighest value in the current heatmap, removes the disk around it, tillthe items are removed from the heatmap.

In the ordered list heatmap, each item has the knowledge of theproceeding item, and the following item. When removing an item from theordered list, we make the following changes, as illustrated in FIG.17-B:

-   -   Assume the removing item is x_(r), its proceeding item is x_(p),        and its following item is x_(f).    -   For the proceeding item x_(p), re-define its following item to        the following item of the removing item. Thus, the following        item of x_(p) is now x_(f).    -   For the removing item x_(r), un-define its proceeding item and        following item, which removes it from the ordered list.    -   For the following item x_(f), re-define its proceeding item to        the proceeding item of the removed item. Thus, the proceeding        item of x_(f) is now x_(p).

After all items are removed from the ordered list, the localizationalgorithm is complete. The number of elements in the set loci will bethe count of analytes, and location information is the pixel coordinatefor each s in the set loci.

Another embodiment searches local peak, which is not necessary the onewith the highest heatmap value. To detect each local peak, we start froma random starting point, and search for the local maximal value. Afterwe find the peak, we calculate the local area surrounding the peak butwith smaller value. We remove this region from the heatmap and find thenext peak from the remaining pixels. The process is repeated only allpixels are removed from the heatmap.

Algorithm LocalSearch (s, heatmap) Input: s: starting location (x, y)heatmap Output: s: location of local peak. We only consider pixels ofvalue > 0. Algorithm Cover (s, heatmap) Input: s: location of localpeak. heatmap: Output: cover: a set of pixels covered by peak:

This is a breadth-first-search algorithm starting from s, with onealtered condition of visiting points: a neighbor p of the currentlocation q is only added to cover if heatmap[p]>0 andheatmap[p]<=heatmap[q]. Therefore, each pixel in cover has anon-descending path leading to the local peak s.

Algorithm LocalSearch (s, heatmap) Input: heatmap Output: loci loci ←{}pixels ←{all pixels from heatmap} while pixels is not empty { s ←anypixel from pixels s ←LocalSearch(s, heatmap)   // s is now local peakprobe local region of radius R surrounding s for better local peak r←Cover(s, heatmap) pixels ← pixels \ r // remove all pixels in cover adds to loci

E-3. Example of Present Invention

-   EA1. A method of deep learning for data analysis, comprising:    receiving an image of a test sample, wherein the sample is loaded    into a QMAX device and the image is taken by an imager connected to    the QMAX device, wherein the image includes detectable signals from    an analyte in the test sample; analyzing the image with a detection    model and generating a 2-D data array of the image, wherein the 2-D    data array includes probability data of the analyte for each    location in the image, and the detection model is established    through a training process that comprises: feeding an annotated data    set to a convolutional neural network, wherein the annotated data    set is from samples that are the same type as the test sample and    for the same analyte; and training and establishing the detection    model by convolution; and analyzing the 2-D data array to detect    local signal peaks with: signal list process, or local searching    process; and calculating the amount of the analyte based on local    signal peak information.-   EB1. A system for data analysis, comprising: a QMAX device, an    imager, and computing unit, wherein: the QMAX device is configured    to compress at least part of a test sample into a layer of highly    uniform thickness; the imager is configured to produce an image of    the sample at the layer of uniform thickness, wherein the image    includes detectable signals from an analyte in the test sample; the    computing unit is configured to: receive the image from the imager;    analyze the image with a detection model and generate a 2-D data    array of the image, wherein the 2-D data array includes probability    data of the analyte for each location in the image, and the    detection model is established through a training process that    comprises: feeding an annotated data set to a convolutional neural    network, wherein the annotated data set is from samples that are the    same type as the test sample and for the same analyte; and training    and establishing the detection model by convolution; and analyzing    the 2-D data array to detect local signal peaks with signal list    process, or local searching process; and calculate the amount of the    analyte based on local signal peak information.-   EA2. The method of embodiment EA1, wherein the signal list process    comprises:

establishing a signal list by iteratively detecting local peaks from the2-D data array, calculating a local area surrounding the detected localpeak, and removing the detected peak and the local area data into thesignal list in order; and sequentially and repetitively removing highestsignals from the signal list and signals from around the highest signal,thus detecting local signal peaks.

-   EA3. The method of any embodiments EA embodiments, wherein the local    search process comprises:    -   i. searching for a local maximal value in the 2-D data array by        starting from a random point;    -   ii. calculating the local area surrounding the peak but with        smaller value;    -   iii. removing the local maximal value and the surrounding        smaller values from the 2-D data array; and    -   iv. repeating steps i-iii to detect local signal peaks.-   EA4. The method of any prior EA embodiments, wherein the annotated    data set is partitioned before annotation.-   EB2. The system of embodiment EB1, wherein the imager comprises a    camera.-   EB3. The system of embodiment EB2, wherein the camera is part of a    mobile communication device.-   EB4. The system of any prior EB embodiments, wherein the computing    unit is part of a mobile communication device.

F. Devices and Methods for Tissue Staining and Cell Imaging F-1. Exampleof QMAX Device for Tissue Staining and Cell Imaging

FIG. 18-A shows an embodiment of a generic QMAX device, that have or nothave a hinge, and wherein Q: quantification; M: magnifying; A: addingreagents; X: acceleration; also known as compressed regulated open flow(CROF)) device. The generic QMAX device comprises a first plate 10 and asecond plate 20. In particular, panel (A) shows the perspective view ofa first plate 10 and a second plate 20 wherein the first plate hasspacers. It should be noted, however, that the spacers also are fixed onthe second plate 20 (not shown) or on both first plate 10 and secondplate 20 (not shown). Panel (B) shows the perspective view and asectional view of depositing a sample 90 on the first plate 10 at anopen configuration. It should be noted, however, that the sample 90 alsois deposited on the second plate 20 (not shown), or on both the firstplate 10 and the second plate 20 (not shown). Panel (C) illustrates (i)using the first plate 10 and second plate 20 to spread the sample 90(the sample flow between the inner surfaces of the plates) and reducethe sample thickness, and (ii) using the spacers and the plate toregulate the sample thickness at the closed configuration of the QMAXdevice. The inner surfaces of each plate have one or a plurality ofbinding sites and or storage sites (not shown).

In some embodiments, the spacers 40 have a predetermined uniform heightand a predetermined uniform inter-spacer distance. In the closedconfiguration, as shown in panel (C) of FIG. 18-A, the spacing betweenthe plates and the thus the thickness of the sample 90 is regulated bythe spacers 40. In some embodiments, the uniform thickness of the sample90 is substantially similar to the uniform height of the spacers 40. Itshould be noted that although FIG. 18-A shows the spacers 40 to be fixedon one of the plates, in some embodiments the spacers are not fixed. Forexample, in certain embodiments the spacers is mixed with the sample sothat when the sample is compressed into a thin layer, the spacers, whichis rigid beads or particles that have a uniform size, regulate thethickness of the sample layer.

FIG. 18-A shows an embodiment of a QMAX device used for cell imaging. Asshown in the figure, the device comprises a first plate 10, a secondplate 20, and spacers 40. The plates are movable relative to each otherinto different configurations, one or both plates are flexible. Each ofthe plates has, on its respective inner surface, a sample contact area(not indicated) for contacting a staining liquid 910 and/or a tissuesample 90 suspected of containing a target analyte. The second plate 20comprises the spacers 40 that are fixed to its inner surface 21. Thespacers 40 have a predetermined substantially uniform height and apredetermined inter-spacer distance, and at least one of the spacers isinside the sample contact area.

FIG. 18-A panels (A) and (B) illustrate one of the configurations, anopen configuration. As shown in the figure, in the open configuration,the two plates are partially or entirely separated apart, the spacing102 between the plates is not regulated by the spacers 40, and thestaining liquid 910 and the sample 90 are deposited on the first plate10. It should be noted, the staining liquid 910 and the sample 90 canalso be deposited on the second plate 20 or both plates.

FIG. 18-A panel (C) depicts another of the configurations of the twoplates, a closed configuration. The closed configuration is configuredafter the deposition of the staining liquid 910 and the sample 90 in theopen configuration, as shown in panel (B). And in the closedconfiguration, at least part of the sample 90 is between the two platesand a layer of at least part of staining liquid 910 is between the atleast part of the sample 90 and the second plate 20, wherein thethickness of the at least part of staining liquid layer is regulated bythe plates, the sample 90, and the spacers 40, and has an averagedistance between the sample surface and the second plate surface isequal or less than 250 μm with a small variation.

In some embodiments, the sample can dried thereon at the openconfiguration, and wherein the sample comprises bodily fluid selectedfrom the group consisting of: amniotic fluid, aqueous humour, vitreoushumour, blood (e.g., whole blood, fractionated blood, plasma or serum),breast milk, cerebrospinal fluid (CSF), cerumen (earwax), chyle, chime,endolymph, perilymph, feces, breath, gastric acid, gastric juice, lymph,mucus (including nasal drainage and phlegm), pericardial fluid,peritoneal fluid, pleural fluid, pus, rheum, saliva, exhaled breathcondensates, sebum, semen, sputum, sweat, synovial fluid, tears, vomit,urine, and any combination thereof.

In some embodiments, the sample contact area of one or both of theplates is configured such that the sample can dried thereon at the openconfiguration, and the sample comprises blood smear and is dried on oneor both plates.

In some embodiments, the sample is a solid tissue section having athickness in the range of 1-200 um, and the sample contact area of oneor both of the plates is adhesive to the sample. In some embodiments,the sample is paraffin-embedded. In some embodiments, the sample isfixed.

In some embodiments, the staining liquid is a pure buffer solution thatdoes not comprise particularly component capable of altering theproperties of the sample. In some embodiments, the staining liquidcomprises fixative capable of fixing the sample. In some embodiments,the staining liquid comprises blocking agents, wherein the blockingagents are configured to disable non-specific endogenous species in thesample to react with detection agents that are used to specificallylabel the target analyte. In some embodiments, the staining liquidcomprises deparaffinizing agents capable of removing paraffin in thesample. In some embodiments, the staining liquid comprisespermeabilizing agents capable of permeabilizing cells in the tissuesample that contain the target analyte. In some embodiments, thestaining liquid comprises antigen retrieval agents capable offacilitating retrieval of antigen. In some embodiments, the stainingliquid comprises detection agents that specifically label the targetanalyte in the sample.

In some embodiments, the sample contact area of one or both platescomprise a storage site that contains blocking agents, wherein theblocking agents are configured to disable non-specific endogenousspecies in the sample to react with detection agents that are used tospecifically label the target analyte. In some embodiments, the samplecontact area of one or both plates comprise a storage site that containsdeparaffinizing agents capable of removing paraffin in the sample. Insome embodiments. the sample contact area of one or both plates comprisea storage site that contains permeabilizing agents capable ofpermeabilizing cells in the tissue sample that contain the targetanalyte. In some embodiments. the sample contact area of one or bothplates comprise a storage site that contains antigen retrieval agentscapable of facilitating retrieval of antigen. In some embodiments, thesample contact area of one or both plates comprise a storage site thatcontains detection agents that specifically label the target analyte inthe sample. In some embodiments, the sample contact area of one or bothof the plates comprise a binding site that contains capture agents,wherein the capture agents are configured to bind to the target analyteon the surface of cells in the sample and immobilize the cells.

In some embodiments, the detection agent comprises dyes for a stainselected from the group consisting of: Acid fuchsin, Alcian blue 8 GX,Alizarin red S, Aniline blue WS, Auramine O, Azocarmine B, Azocarmine G,Azure A, Azure B, Azure C, Basic fuchsine, Bismarck brown Y, Brilliantcresyl blue, Brilliant green, Carmine, Chlorazol black E, Congo red,C.I. Cresyl violet, Crystal violet, Darrow red, Eosin B, Eosin Y,Erythrosin, Ethyl eosin, Ethyl green, Fast green F C F, FluoresceinIsothiocyanate, Giemsa Stain, Hematoxylin, Hematoxylin & Eosin, Indigocarmine, Janus green B, Jenner stain 1899, Light green SF, Malachitegreen, Martius yellow, Methyl orange, Methyl violet 2B, Methylene blue,Methylene blue, Methylene violet, (Bernthsen), Neutral red, Nigrosin,Nile blue A, Nuclear fast red, Oil Red, Orange G, Orange II, Orcein,Pararosaniline, Phloxin B, Protargol S, Pyronine B, Pyronine, Resazurin,Rose Bengal, Safranine O, Sudan black B, Sudan III, Sudan IV,Tetrachrome stain (MacNeal), Thionine, Toluidine blue, Weigert, Wrightstain, and any combination thereof.

In some embodiments, the detection agent comprises antibodies configuredto specifically bind to protein analyte in the sample.

In some embodiments, the detection agent comprises oligonucleotideprobes configured to specifically bind to DNA and/or RNA in the sample.

In some embodiments, the detection agent is labeled with a reportermolecule, wherein the reporter molecule is configured to provide adetectable signal to be read and analyzed.

In some embodiments, the signal is selected from the group consistingof:

-   -   i. luminescence selected from photoluminescence,        electroluminescence, and electrochemiluminescence;    -   ii. light absorption, reflection, transmission, diffraction,        scattering, or diffusion;    -   iii. surface Raman scattering;    -   iv. electrical impedance selected from resistance, capacitance,        and inductance;    -   v. magnetic relaxivity; and    -   vi. any combination of i-v.

F-2. Immunohistochemistry

In some embodiments, the devices and methods of the present inventionare useful for conducting immunohistochemistry on the sample.

In immunohistochemical (IHC) staining methods, a tissue sample is fixed(e.g., in paraformaldehyde), optionally embedding in wax, sliced intothin sections that are less then 100 um thick (e.g., 2 um to 6 umthick), and then mounted onto a support such as a glass slide. Oncemounted, the tissue sections may be dehydrated using alcohol washes ofincreasing concentrations and cleared using a detergent such as xylene.

In most IHC methods, a primary and a secondary antibody may be used. Insuch methods, the primary antibody binds to antigen of interest (e.g., abiomarker) and is unlabeled. The secondary antibody binds to the primaryantibody and directly conjugated either to a reporter molecule or to alinker molecule (e.g., biotin) that can recruit reporter molecule thatis in solution. Alternatively, the primary antibody itself may bedirectly conjugated either to a reporter molecule or to a linkermolecule (e.g., biotin) that can recruit reporter molecule that is insolution. Reporter molecules include fluorophores (e.g., FITC, TRITC,AMCA, fluorescein and rhodamine) and enzymes such as alkalinephosphatase (AP) and horseradish peroxidase (HRP), for which there are avariety of fluorogenic, chromogenic and chemiluminescent substrates suchas DAB or BCIP/NBT.

In direct methods, the tissue section is incubated with a labeledprimary antibody (e.g. an FITC-conjugated antibody) in binding buffer.The primary antibody binds directly with the antigen in the tissuesection and, after the tissue section has been washed to remove anyunbound primary antibody, the section is to be analyzed by microscopy.

In indirect methods, the tissue section is incubated with an unlabeledprimary antibody that binds to the target antigen in the tissue. Afterthe tissue section is washed to remove unbound primary antibody, thetissue section is incubated with a labeled secondary antibody that bindsto the primary antibody.

After immunohistochemical staining of the antigen, the tissue sample maybe stained with another dye, e.g., hematoxylin, Hoechst stain and DAPI,to provide contrast and/or identify other features.

The present device may be used for immunohistochemical (IHC) staining atissue sample. In these embodiments, the device may comprise a firstplate and a second plate, wherein: the plates are movable relative toeach other into different configurations; one or both plates areflexible; each of the plates has, on its respective surface, a samplecontact area for contacting a tissue sample or a IHC staining liquid;the sample contact area in the first plate is smooth and planner; thesample contact area in the second plate comprise spacers that are fixedon the surface and have a predetermined substantially uniform height anda predetermined constant inter-spacer distance that is in the range of 7μm to 200 μm;

wherein one of the configurations is an open configuration, in which:the two plates are completely or partially separated apart, the spacingbetween the plates is not regulated by the spacers; and wherein anotherof the configurations is a closed configuration which is configuredafter a deposition of the sample and the IHC staining liquid in the openconfiguration; and in the closed configuration: at least part of thesample is between the two plates and a layer of at least part ofstaining liquid is between the at least part of the sample and thesecond plate, wherein the thickness of the at least part of stainingliquid layer is regulated by the plates, the sample, and the spacers,and has an average distance between the sample surface and the secondplate surface is equal or less than 250 μm with a small variation.

In some embodiments, the device may comprise a dry IHC staining agentcoated on the sample contact area of one or both plates. In someembodiments, the device may comprise a dry IHC staining agent coated onthe sample contact area of the second plate, and the IHC staining liquidcomprise a liquid that dissolve the dry IHC staining agent. The deviceof claim 1, wherein the thickness of the sample is 2 um to 6 um.

F-3. H& E and Special Stains

In some embodiments, the devices and methods of the present inventionare useful for conducting H&E stain and special stains.

Hematoxylin and eosin stain or haematoxylin and eosin stain (H&E stainor HE stain) is one of the principal stains in histology. It is the mostwidely used stain in medical diagnosis and is often the gold standard;for example when a pathologist looks at a biopsy of a suspected cancer,the histological section is likely to be stained with H&E and termed“H&E section”, “H+E section”, or “HE section”. A combination ofhematoxylin and eosin, it produces blues, violets, and reds.

In diagnostic pathology, the “special stain” terminology is mostcommonly used in the clinical environment, and simply means anytechnique other than the H & E method that is used to impart colors to aspecimen. This also includes immunohistochemical and in situhybridization stains. On the other hand, the H & E stain is the mostpopular staining method in histology and medical diagnosis laboratories.

In any embodiments, the dry binding site may comprise a capture agentsuch as an antibody or nucleic acid. In some embodiments, the releasabledry reagent may be a labeled reagent such as a fluorescently-labeledreagent, e.g., a fluorescently-labeled antibody or a cell stain suchRomanowsky's stain, Leishman stain, May-Grunwald stain, Giemsa stain,Jenner's stain, Wright's stain, or any combination of the same (e.g.,Wright-Giemsa stain). Such a stain may comprise eosin Y or eosin B withmethylene blue. In certain embodiments, the stain may be an alkalinestain such as haematoxylin.

In some embodiments, the special stains include, but not limited to,Acid fuchsin, Alcian blue 8 GX, Alizarin red S, Aniline blue WS,Auramine O, Azocarmine B, Azocarmine G, Azure A, Azure B, Azure C, Basicfuchsine, Bismarck brown Y, Brilliant cresyl blue, Brilliant green,Carmine, Chlorazol black E, Congo red, C.I. Cresyl violet, Crystalviolet, Darrow red, Eosin B, Eosin Y, Erythrosin, Ethyl eosin, Ethylgreen, Fast green F C F, Fluorescein Isothiocyanate, Giemsa Stain,Hematoxylin, Hematoxylin & Eosin, Indigo carmine, Janus green B, Jennerstain 1899, Light green SF, Malachite green, Martius yellow, Methylorange, Methyl violet 2B, Methylene blue, Methylene blue, Methyleneviolet, (Bernthsen), Neutral red, Nigrosin, Nile blue A, Nuclear fastred, Oil Red, Orange G, Orange II, Orcein, Pararosaniline, Phloxin B,Protargol S, Pyronine B, Pyronine, Resazurin, Rose Bengal, Safranine O,Sudan black B, Sudan III, Sudan IV, Tetrachrome stain (MacNeal),Thionine, Toluidine blue, Weigert, Wright stain, and any combinationthereof.

F-4. In Situ Hybridization

In some embodiments, the devices and methods of the present inventionare useful for conducting in situ hybridization (ISH) on histologicalsamples.

In situ hybridization (ISH) is a type of hybridization that uses alabeled complementary DNA, RNA or modified nucleic acids strand (i.e.,probe) to localize a specific DNA or RNA sequence in a portion orsection of tissue (in situ), or, if the tissue is small enough (e.g.,plant seeds, Drosophila embryos), in the entire tissue (whole mountISH), in cells, and in circulating tumor cells (CTCs).

In situ hybridization is used to reveal the location of specific nucleicacid sequences on chromosomes or in tissues, a crucial step forunderstanding the organization, regulation, and function of genes. Thekey techniques currently in use include: in situ hybridization to mRNAwith oligonucleotide and RNA probes (both radio-labelled andhapten-labelled); analysis with light and electron microscopes; wholemount in situ hybridization; double detection of RNAs and RNA plusprotein; and fluorescent in situ hybridization to detect chromosomalsequences. DNA ISH can be used to determine the structure ofchromosomes. Fluorescent DNA ISH (FISH) can, for example, be used inmedical diagnostics to assess chromosomal integrity. RNA ISH (RNA insitu hybridization) is used to measure and localize RNAs (mRNAs,IncRNAs, and miRNAs) within tissue sections, cells, whole mounts, andcirculating tumor cells (CTCs).

In some embodiments, the detection agent comprises nucleic acid probesfor in situ hybridization staining. The nucleic acid probes include, butnot limited to, oligonucleotide probes configured to specifically bindto DNA and/or RNA in the sample.

F-5. System and Method for Tissue Staining and Cell Imaging

Also provided is a system for rapidly staining and analyzing a tissuesample using a mobile phone comprising:

(a) sample, staining liquid, and device as described above,

(b) a mobile communication

device comprising:

i. one or a plurality of cameras for the detecting and/or imaging thesample;

ii. electronics, signal processors, hardware and software for receivingand/or processing the detected signal and/or the image of the sample andfor remote communication; and

(c) a light source from either the mobile communication device or anexternal source.

Also provided is a method for rapidly staining and analyzing a tissuesample using a mobile phone, comprising:

(a) depositing a tissue sample and a staining liquid on the device ofthe system described above, and placing the two plate into a closedconfiguration;

(b) obtaining a mobile phone that has hardware and software of imaging,data processing, and communication;

(c) assaying by the tissue sample deposited on the CROF device by themobile phone to generate a result; and

(d) communicating the result from the mobile phone to a location remotefrom the mobile phone.

Also provided is a method for staining a tissue sample, comprising:

(a) obtaining a tissue sample;

(b) obtaining a stain liquid;

(c) obtaining a first plate and a second plate, wherein:

the plates are movable relative to each other into differentconfigurations;

one or both plates are flexible;

each of the plates has, on its respective surface, a sample contact areafor contacting a tissue sample or a IHC staining liquid;

the sample contact area in the first plate is smooth 5 and planner;

the sample contact area in the second plate comprise spacers that arefixed on the surface and have a predetermined substantially uniformheight and a predetermined constant inter-spacer distance that is in therange of 7 μm to 200 μm;

(c) depositing the tissue sample and the stain liquid on the plates whenthe plates are configured in an open configuration, wherein the openconfiguration is a configuration in which the two plates are eitherpartially or completely separated apart and the spacing between theplates is not regulated by the spacers; and

(d), after (c), using the two plates to compress at least part of thetissue sample and at least part of the staining liquid into a closedconfiguration;

wherein in the closed configuration: at least part of the sample isbetween the two plates and a layer of at least part of staining liquidis between the at least part of the sample and the second plate, whereinthe thickness of the at least part of staining liquid layer is regulatedby the plates, the sample, and the spacers, and has an average distancebetween the sample surface and the second plate surface is equal or lessthan 250 μm with a small variation.

All of the benefits and advantages (e.g., an accelerated reaction,faster results, etc.) of other embodiments may be applied to thisdevice, system and method.

Further, all parameters described above in the context of otherembodiments (e.g., the size, spacing and shape of the spacers, theflexibility of the spacers and plates, and how the device and system canbe used, etc.) can be incorporated into IHC embodiments described inthis section.

For example, in some embodiments, the spacers regulating the layer ofuniform thickness (i.e., the spacers that are spacing the plates awayfrom each other in the layer) have a “filling factor” of at least 1%,e.g., at least 2% or at least 5%, wherein the filling factor is theratio of the spacer area that is in contact with the layer of uniformthickness to the total plate area that is in contact with the layer ofuniform thickness. In some embodiments, for spacers regulating the layerof uniform thickness, the Young's modulus of the spacers times thefilling factor of the spacers is equal or larger than 10 MPa, e.g., atleast 15 MPa or at least 20 MPa, where the filling factor is the ratioof the spacer area that is in contact with the layer of uniformthickness to the total plate area that is in contact with the layer ofuniform thickness. In some embodiments, the thickness of the flexibleplate times the Young's modulus of the flexible plate is in the range of60 to 550 GPa-um, e.g., 100 to 300 GPa-um. In some embodiments, for aflexible plate, the fourth power of the inter-spacer-distance (ISD)divided by the thickness of the flexible plate (h) and the Young'smodulus (E) of the flexible plate, ISD⁴/(hE), is equal to 5 or less than10⁶ um³/GPa, e.g., less than 10⁵ um³/GPa, less than 10⁴ um³/GPa or lessthan 10³ um³/GPa.

In some embodiments, one or both plates comprise a location markereither on a surface of or inside the plate, that provide information ofa location of the plate, e.g., a location that is going to be analyzedor a location onto which the section should be deposited. In some cases,one or both plates may comprise a scale marker, either on a surface ofor inside the plate, that provides information of a lateral dimension ofa structure of the section and/or the plate. In some embodiments, one orboth plates comprise an imaging marker, either on surface of or insidethe plate, that assists an imaging of the sample. For example, theimaging marker could help focus the imaging device or direct the imagingdevice to a location on the device. In some embodiments, the spacers canfunction as a location marker, a scale marker, an imaging marker, or anycombination of thereof.

In some embodiments, the inter-spacer distance may substantiallyperiodic. In some cases, the spacers may be in a regular pattern and thespacing between adjacent spacers may be approximately the same. In someembodiments, the spacers are pillars with a cross-sectional shapeselected from round, polygonal, circular, square, rectangular, oval,elliptical, or any combination of the same and, in some embodiments, thespacers may have a substantially flat top surface, wherein, for eachspacer, the ratio of the lateral dimension of the spacer to its heightis at least 1. In some cases, the minimum lateral dimension of spacer isless than or substantially equal to the minimum dimension of an analytein the sample. The minimum lateral dimension of spacer is in the rangeof 0.5 um to 100 um, e.g., in the range of 2 um to 50 um or 0.5 um to 10um.

In some embodiments, the spacers have a pillar shape and the sidewallcorners of the spacers have a round shape with a radius of curvature atleast 1 um, e.g., at least 1.2 um, at least 1.5 um or at least 2.0 um.The spacers may have any convenient density, e.g., a density of at least1000/mm², e.g., a density of at least 1000/mm², a density of at least2000/mm², a density of at least 5,000/mm² or a density of at least10,000/mm².

In this device, at least one of the plates may be transparent, therebyallowing the assay to be read optically. Likewise, in this device, atleast one of the plates may be made of a flexible polymer, therebyallowing the sample to be efficiently spread by compressing the platestogether. In some embodiments, the pressure that compresses the plates,the spacers are not compressible and/or, independently, only one of theplates is flexible. The flexible plate may have a thickness in the rangeof 20 um to 200 um, e.g., 50 um to 150 um. As noted above, in the closedposition, the thickness of the layer of uniform thickness may have asmall variation.

In some embodiments, the variation may be less than 10%, less than 5% orless than 2%, meaning that the thickness of the area does not exceed+/−10%, +/−5% 5 or +/−2% of the average thickness.

In some embodiments, the first and second plates are connected and thedevice can be changed from the open configuration to the closedconfiguration by folding the plates. In some embodiments, the first andsecond plates can be connected by a hinge and the device can be changedfrom the open configuration to the closed configuration by folding theplates such that the device bends along the hinge. The hinge may be aseparate material that is attached to the plates or, in some cases, theplates may be integral with the plates.

In some embodiments, the device may be capable of analyzing the sectionvery rapidly. In some cases, the analysis may be done in 60 seconds orless, in 30 seconds, in 20 seconds or 15 less or in 10 seconds or less.

In some embodiments, the system may additionally comprise (d) a housingconfigured to hold the sample and to be mounted to the mobilecommunication device. The housing may comprise optics for facilitatingthe imaging and/or signal processing of the sample by the mobilecommunication device, and a mount configured to hold the optics on themobile communication device. In some cases, an element of the optics ofthe device (e.g., a lens, filter, mirror, prism or a beamsplitter, maybe movable) such that the sample may be imaged in at least two channels.

In some embodiments, the mobile communication device may be configuredto communicate test results to a medical professional (e.g., an MD), amedical facility (e.g., a hospital or testing lab) or an insurancecompany. In addition, the mobile communication device may be configuredto communicate information on the subject (e.g., the subject's age,gender, weight, address, name, prior test results, prior medicalhistory, etc.) with the medical professional, medical facility orinsurance company. In certain embodiments, the mobile communicationdevice may be configured to receive a prescription, diagnosis or arecommendation from a medical professional. For example, in someembodiments the mobile communication device may send assay results to aremote location where a medical professional gives a diagnosis. Thediagnosis may be communicated to the subject via the mobilecommunication device.

In some embodiments, the mobile communication device may containhardware and software that allows it to (a) capture an image of thesample; (b) analyze a test location and a control location in in image;and (c) compare a value obtained from analysis of the test location to athreshold value that characterizes the rapid diagnostic test. In somecases, the mobile communication device communicates with the remotelocation via a wireless or cellular network.

In any embodiment, the mobile communication device may be a mobilephone.

The system may be used in a method that comprises (a) sample on thedevice of the system; (b) assaying the sample deposited on the device togenerate a result; and (c) communicating the result from the mobilecommunication device to a location remote from the mobile communicationdevice. The method may comprise analyzing the results at the remotelocation to provide an analyzed result; and communicating the analyzedresult from the remote location to the mobile communication device. Asnoted above, the analysis may be done by a medical professional at aremote location. And, in some embodiments, the mobile communicationdevice may receive a prescription, diagnosis or a recommendation from amedical professional at a remote location.

Also provided is a method for analyzing a tissue section. In someembodiments, this method may comprise obtaining a device as describedabove, depositing the section onto one or both pates of the device;placing the plates in a closed configuration and applying an externalforce over at least part of the plates; and analyzing the sample in thelayer of uniform thickness while the plates are the closedconfiguration.

In some embodiments, this method may comprise:

(a) obtaining a tissue section;

(b) obtaining a first and second plates that are movable relative toeach other into different configurations, wherein each plate has asample contact surface that is substantially planar, one or both platesare flexible, and one or both of the plates comprise spacers that arefixed with a respective sample contacting surface, and wherein thespacers have:

i. a predetermined substantially uniform height,

ii. a shape of pillar with substantially uniform cross-section and aflat top surface;

iii. a ratio of the width to the height equal or larger than one;

iv. a predetermined constant inter-spacer distance that is in the rangeof 10 μm to 200 μm;

v. a filling factor of equal to 1% or larger; and

(c) depositing the section on one or both of the plates when the platesare configured in an open configuration, wherein the open configurationis a configuration in which 5 the two plates are either partially orcompletely separated apart and the spacing between the plates is notregulated by the spacers;

(d), after (c), using the two plates to compress at least part of thesection into a layer of substantially uniform thickness that is confinedby the sample contact surfaces of the plates, wherein the uniformthickness of the layer is regulated by the spacers and the plates, andhas an average value in the range of 1.8 μm to 3 μm with a variation ofless than 10%, wherein the compressing comprises:

bringing the two plates together; and

conformable pressing, either in parallel or sequentially, an area of atleast one of the plates to press the plates together to a closedconfiguration, wherein the conformable pressing generates asubstantially uniform pressure on the plates over the at least part ofthe sample, and the pressing spreads the at least part of the samplelaterally between the sample contact surfaces of the plates, and whereinthe closed configuration is a configuration in which the spacing betweenthe plates in the layer of uniform thickness region is regulated by thespacers; and

(e) analyzing the section in the layer of uniform thickness while theplates are the closed configuration;

wherein the filling factor is the ratio of the spacer contact area tothe total plate area;

wherein a conformable pressing is a method that makes the pressureapplied over an area is substantially constant regardless the shapevariation of the outer surfaces of the plates;

and

wherein the parallel pressing applies the pressures on the intended areaat the same time, and a sequential pressing applies the pressure on apart of the intended area and gradually move to other area.

In some embodiments, this method may comprise: removing the externalforce after the plates are in the closed configuration; imaging thesection in the layer of uniform thickness while the plates are theclosed configuration. As noted above, in these embodiments, theinter-spacer distance may in the range of 20 um to 200 um or 5 um to 20um. In these embodiments, the product of the filling factor and theYoung's modulus of the spacer is 2 MPa or larger. In some embodiments,the surface variation is less than 30 nm.

In any of these embodiments, the imaging and counting may be done by: i.illuminating the section in the layer of uniform thickness; ii. takingone or more images of the section using a CCD or CMOS sensor.

In some embodiments, the external force may be provided by human 5 hand,e.g., by pressing down using a digit such as a thumb, or pinchingbetween a thumb and another digit such as a forefinger on the same hand.

In some embodiments, one or more of the plates may comprises a dryreagent coated on one or both plates (e.g., a binding agent, a stainingagent, a detection agent or an assay reactant).

In some embodiments, the layer of uniform thickness sample may athickness uniformity of up to +/−5%, e.g., up to +/−2% or up to +/−1%.

In some embodiments, the spacers are pillars with a cross-sectionalshape selected from round, polygonal, circular, square, rectangular,oval, elliptical, or any combination of the same.

F-6. Examples of Present Invention

-   FA1. A device for analyzing a tissue sample, comprising: a first    plate, a second plate, and spacers, wherein: the plates are movable    relative to each other into different configurations; one or both    plates are flexible; each of the plates has, on its respective inner    surface, a sample contact area for contacting a staining liquid    and/or a tissue sample suspected of containing a target analyte; one    or both of the plates comprise the spacers that are fixed with a    respective plate; the spacers have a predetermined substantially    uniform height and a predetermined inter-spacer distance; and at    least one of the spacers is inside the sample contact area; wherein    one of the configurations is an open configuration, in which: the    two plates are partially or entirely separated apart, the spacing    between the plates is not regulated by the spacers, and the staining    liquid and the sample are deposited on one or both of the plates;    wherein another of the configurations is a closed configuration,    which is configured after the deposition of the staining liquid and    the sample in the open configuration, and in the closed    configuration: at least part of the sample is between the two plates    and a layer of at least part of staining liquid is between the at    least part of the sample and the second plate, wherein the thickness    of the at least part of staining liquid layer is regulated by the    plates, the sample, and the spacers, and has an average distance    between the sample surface and the second plate surface is equal or    less than 250 μm with a small variation.-   FAA1. A device for analyzing a tissue sample, comprising: a first    plate, a second plate, and spacers, wherein: the plates are movable    relative to each other into different configurations; one or both    plates are flexible; each of the plates has, on its respective inner    surface, a sample contact area for contacting a transfer solution    and/or a tissue sample suspected of containing a target analyte; one    or both of the plates comprise stain agent that is dried on the    respective sample contact area and configured to, upon contacting    the transfer solution, dissolve in the transfer solution and stain    the tissue sample; one or both of the plates comprise the spacers    that are fixed with a respective plate; the spacers have a    predetermined substantially uniform height and a predetermined    inter-spacer distance; and at least one of the spacers is inside the    sample contact area; wherein one of the configurations is an open    configuration, in which: the two plates are partially or entirely    separated apart, the spacing between the plates is not regulated by    the spacers, and the staining liquid and the sample are deposited on    one or both of the plates; wherein another of the configurations is    a closed configuration, which is configured after the deposition of    the staining liquid and the sample in the open configuration, and in    the closed configuration: at least part of the sample is between the    two plates and a layer of at least part of transfer solution is    between the at least part of the sample and the second plate,    wherein the thickness of the at least part of transfer solution    layer is regulated by the plates, the sample, and the spacers, and    has an average distance between the sample surface and the second    plate surface is equal or less than 250 μm with a small variation.-   FB1. A method for analyzing a tissue sample, comprising the steps    of:

(a) obtaining a tissue sample suspected of containing a target analyteand a staining liquid;

(b) obtaining a first plate, a second plate, and spacers, wherein:

-   -   i. the plates are movable relative to each other into different        configurations;    -   ii. one or both plates are flexible;    -   iii. each of the plates has, on its respective inner surface, a        sample contact area for contacting the staining liquid and/or        the tissue sample;    -   iv. one or both of the plates comprise the spacers that are        fixed with a respective plate;    -   v. the spacers have a predetermined substantially uniform height        and a predetermined inter-spacer distance; and    -   vi. at least one of the spacers is inside the sample contact        area;

(c) depositing the staining liquid and the tissue sample on one or bothof the plates when the plates are in an open configuration,

-   -   wherein the open configuration is a configuration in which the        two plates are partially or entirely separated apart, the        spacing between the two plates is not regulated by the spacers,        and the sample and the staining liquid are deposited on one or        both of the plates;

(d) after (c), bringing the two plates together and pressing the platesinto a closed configuration,

-   -   wherein the pressing comprises conformable pressing, either in        parallel or sequentially, an area of at least one of the plates        to press the plates together to the closed configuration,        wherein the conformable pressing generates a substantially        uniform pressure on the plates over the at least part of the        sample, and the pressing spreads the at least part of the sample        laterally between the inner surfaces of the plates; and    -   wherein another of the configurations is the closed        configuration, which is configured after the deposition of the        staining liquid and the sample in the open configuration, and in        the closed configuration: at least part of the sample is between        the two plates and a layer of at least part of staining liquid        is between the at least part of the sample and the second plate,        wherein the thickness of the at least part of staining liquid        layer is regulated by the plates, the sample, and the spacers,        and has an average distance between the sample surface and the        second plate surface is equal or less than 250 μm with a small        variation; and

(e) analyzing the target analyte when the plates are in the closedconfiguration.

-   FBB1. A method for analyzing a tissue sample, comprising the steps    of:

(a) obtaining a tissue sample suspected of containing a target analyteand a transfer solution;

(b) obtaining a first plate, a second plate, and spacers, wherein:

-   -   i. the plates are movable relative to each other into different        configurations;    -   ii. one or both plates are flexible;    -   iii. each of the plates has, on its respective inner surface, a        sample contact area for contacting a staining liquid and/or a        tissue sample suspected of containing a target analyte;    -   iv. one or both of the plates comprise stain agents that are        coated on the respective sample contact area and configured to,        upon contacting a transfer solution, dissolve in the transfer        solution and stain the tissue sample;    -   v. one or both of the plates comprise the spacers that are fixed        with a respective plate;    -   vi. the spacers have a predetermined substantially uniform        height and a predetermined inter-spacer distance; and    -   vii. at least one of the spacers is inside the sample contact        area;

(c) depositing the staining liquid and the tissue sample on one or bothof the plates when the plates are in an open configuration,

-   -   wherein the open configuration is a configuration in which the        two plates are partially or entirely separated apart, the        spacing between the two plates is not regulated by the spacers,        and the sample and the staining liquid are deposited on one or        both of the plates;

(d) after (c), bringing the two plates together and pressing the platesinto a closed configuration,

-   -   wherein the pressing comprises conformable pressing, either in        parallel or sequentially, an area of at least one of the plates        to press the plates together to the closed configuration,        wherein the conformable pressing generates a substantially        uniform pressure on the plates over the at least part of the        sample, and the pressing spreads the at least part of the sample        laterally between the inner surfaces of the plates; and    -   wherein another of the configurations is the closed        configuration, which is configured after the deposition of the        staining liquid and the sample in the open configuration, and in        the closed configuration: at least part of the sample is between        the two plates and a layer of at least part of staining liquid        is between the at least part of the sample and the second plate,        wherein the thickness of the at least part of staining liquid        layer is regulated by the plates, the sample, and the spacers,        and has an average distance between the sample surface and the        second plate surface is equal or less than 250 μm with a small        variation;

and

(e) analyzing the target analyte when the plates are in the closedconfiguration.

-   FA2. The device of embodiment FA1, wherein one or both of the plates    is configured such that the sample can dried thereon at the open    configuration, and wherein the sample comprises bodily fluid    selected from the group consisting of: amniotic fluid, aqueous    humour, vitreous humour, blood (e.g., whole blood, fractionated    blood, plasma or serum), breast milk, cerebrospinal fluid (CSF),    cerumen (earwax), chyle, chime, endolymph, perilymph, feces, breath,    gastric acid, gastric juice, lymph, mucus (including nasal drainage    and phlegm), pericardial fluid, peritoneal fluid, pleural fluid,    pus, rheum, saliva, exhaled breath condensates, sebum, semen,    sputum, sweat, synovial fluid, tears, vomit, urine, and any    combination thereof.-   FAA2. The device of any prior embodiment, wherein the staining    liquid has a viscosity in the range of 0.1 to 3.5 mPa S.-   FA3. The device of any prior embodiment, wherein the sample contact    area of one or both of the plates is configured such that the sample    can dried thereon at the open configuration, and wherein the sample    comprises blood smear and is dried on one or both plates.-   FA4. The device of any prior embodiment, wherein the sample contact    area of one or both of the plates is adhesive to the sample, and    wherein the sample is a tissue section having a thickness in the    range of 1-200 um.-   FA5. The device of embodiment FA4, wherein the sample is    paraffin-embedded.-   FA6. The device of any of embodiment, wherein the sample is fixed.-   FA7. The device of any prior embodiment, wherein the staining liquid    comprises fixative capable of fixing the sample.-   FA8. The device of any prior embodiment, wherein the staining liquid    comprises blocking agents, wherein the blocking agents are    configured to disable non-specific endogenous species in the sample    to react with detection agents that are used to specifically label    the target analyte.-   FA9. The device of any prior embodiment, wherein the staining liquid    comprises deparaffinizing agents capable of removing paraffin in the    sample.-   FA10. The device of any prior embodiment, wherein the staining    liquid comprises permeabilizing agents capable of permeabilizing    cells in the tissue sample that contain the target analyte.-   FA11. The device of any prior embodiment, wherein the staining    liquid comprises antigen retrieval agents capable of facilitating    retrieval of antigen.-   FA12. The device of any prior embodiment, wherein the staining    liquid comprises detection agents that specifically label the target    analyte in the sample.-   FA13. The device of any prior embodiment, wherein the sample contact    area of one or both plates comprise a storage site that contains    blocking agents, wherein the blocking agents are configured to    disable non-specific endogenous species in the sample to react with    detection agents that are used to specifically label the target    analyte.-   FA14. The device of any prior embodiment, wherein the sample contact    area of one or both plates comprise a storage site that contains    deparaffinizing agents capable of removing paraffin in the sample.-   FA15. The device of any prior embodiment, wherein the sample contact    area of one or both plates comprise a storage site that contains    permeabilizing agents capable of permeabilizing cells in the tissue    sample that contain the target analyte.-   FA16. The device of any prior embodiment, wherein the sample contact    area of one or both plates comprise a storage site that contains    antigen retrieval agents capable of facilitating retrieval of    antigen.-   FA17. The device of any prior embodiment, wherein the sample contact    area of one or both plates comprise a storage site that contains    detection agents that specifically label the target analyte in the    sample.-   FA18. The device of any prior embodiment, wherein the detection    agent comprises dyes for a stain selected from the group consisting    of: Acid fuchsin, Alcian blue 8 GX, Alizarin red S, Aniline blue WS,    Auramine O, Azocarmine B, Azocarmine G, Azure A, Azure B, Azure C,    Basic fuchsine, Bismarck brown Y, Brilliant cresyl blue, Brilliant    green, Carmine, Chlorazol black E, Congo red, C.I. Cresyl violet,    Crystal violet, Darrow red, Eosin B, Eosin Y, Erythrosin, Ethyl    eosin, Ethyl green, Fast green F C F, Fluorescein Isothiocyanate,    Giemsa Stain, Hematoxylin, Hematoxylin & Eosin, Indigo carmine,    Janus green B, Jenner stain 1899, Light green SF, Malachite green,    Martius yellow, Methyl orange, Methyl violet 2B, Methylene blue,    Methylene blue, Methylene violet, (Bernthsen), Neutral red,    Nigrosin, Nile blue A, Nuclear fast red, Oil Red, Orange G, Orange    II, Orcein, Pararosaniline, Phloxin B, Protargol S, Pyronine B,    Pyronine, Resazurin, Rose Bengal, Safranine O, Sudan black B, Sudan    III, Sudan IV, Tetrachrome stain (MacNeal), Thionine, Toluidine    blue, Weigert, Wright stain, and any combination thereof.-   FA19. The device of any prior embodiment, wherein the detection    agent comprises antibodies configured to specifically bind to    protein analyte in the sample.-   FA20. The device of any prior embodiment, wherein the detection    agent comprises oligonucleotide probes configured to specifically    bind to DNA and/or RNA in the sample.-   FA21. The device of any prior embodiment, wherein the detection    agent is labeled with a reporter molecule, wherein the reporter    molecule is configured to provide a detectable signal to be read and    analyzed.-   FA22. The device of embodiment FA21, wherein the signal is selected    from the group consisting of:    -   i. luminescence selected from photoluminescence,        electroluminescence, and electrochemiluminescence;    -   ii. light absorption, reflection, transmission, diffraction,        scattering, or diffusion;    -   iii. surface Raman scattering;    -   iv. electrical impedance selected from resistance, capacitance,        and inductance;    -   v. magnetic relaxivity; and    -   vi. any combination of i-v.-   FA23. The device of any prior embodiment, wherein the sample contact    area of one or both of the plates comprise a binding site that    contains capture agents, wherein the capture agents are configured    to bind to the target analyte on the surface of cells in the sample    and immobilize the cells.-   FB2. The method of embodiment FB1, wherein the depositing step (c)    comprises depositing and drying the sample on one or both of the    plates before depositing the remaining of the staining liquid on top    of the dried sample, and wherein the sample comprises bodily fluid    selected from the group consisting of: amniotic fluid, aqueous    humour, vitreous humour, blood (e.g., whole blood, fractionated    blood, plasma or serum), breast milk, cerebrospinal fluid (CSF),    cerumen (earwax), chyle, chime, endolymph, perilymph, feces, breath,    gastric acid, gastric juice, lymph, mucus (including nasal drainage    and phlegm), pericardial fluid, peritoneal fluid, pleural fluid,    pus, rheum, saliva, exhaled breath condensates, sebum, semen,    sputum, sweat, synovial fluid, tears, vomit, urine, and any    combination thereof.-   FBB2. The method of any prior embodiment, wherein the staining    liquid has a viscosity in the range of 0.1 to 3.5 mPa S.-   FB3. The method of any prior embodiment, wherein the depositing    step (c) comprises depositing and drying the sample on one or both    of the plates before depositing the remaining of the staining liquid    on top of the dried sample, and wherein the sample comprises blood    smear and is dried on one or both plates.-   FB4. The method of any prior embodiment, wherein the depositing    step (c) comprises depositing and attaching the sample to one or    both of the plates before depositing the staining liquid on top of    the sample, wherein the sample contact area of one or both of the    plates is adhesive to the sample, and wherein the sample is a tissue    section having a thickness in the range of 1-200 □m.-   FB5. The device of embodiment FA4, wherein the sample is    paraffin-embedded.-   FB6. The method of any of embodiment, wherein the sample is fixed.-   FB7. The method of any prior embodiment, wherein the staining liquid    comprises fixative capable of fixing the sample.-   FB8. The method of any prior embodiment, wherein the staining liquid    comprises blocking agents, wherein the blocking agents are    configured to disable non-specific endogenous species in the sample    to react with detection agents that are used to specifically label    the target analyte.-   FB9. The method of any prior embodiment, wherein the staining liquid    comprises deparaffinizing agents capable of removing paraffin in the    sample.-   B10. The method of any prior embodiment, wherein the staining liquid    comprises permeabilizing agents capable of permeabilizing cells in    the tissue sample that contain the target analyte.-   FB11. The method of any prior embodiment, wherein the staining    liquid comprises antigen retrieval agents capable of facilitating    retrieval of antigen.-   FB12. The method of any prior embodiment, wherein the staining    liquid comprises detection agents that specifically label the target    analyte in the sample.-   FB13. The method of any prior embodiment, wherein the sample contact    area of one or both plates comprise a storage site that contains    blocking agents, wherein the blocking agents are configured to    disable non-specific endogenous species in the sample to react with    detection agents that are used to specifically label the target    analyte.-   FB14. The method of any prior embodiment, wherein the sample contact    area of one or both plates comprise a storage site that contains    deparaffinizing agents capable of removing paraffin in the sample.-   FB15. The method of any prior embodiment, wherein the sample contact    area of one or both plates comprise a storage site that contains    permeabilizing agents capable of permeabilizing cells in the tissue    sample that contain the target analyte.-   FB16. The method of any prior embodiment, wherein the sample contact    area of one or both plates comprise a storage site that contains    antigen retrieval agents capable of facilitating retrieval of    antigen.-   FB17. The method of any prior embodiment, wherein the sample contact    area of one or both plates comprise a storage site that contains    detection agents that specifically label the target analyte in the    sample.-   FB18. The method of any prior embodiment, wherein the detection    agent comprises dyes for a stain selected from the group consisting    of: Acid fuchsin, Alcian blue 8 GX, Alizarin red S, Aniline blue WS,    Auramine O, Azocarmine B, Azocarmine G, Azure A, Azure B, Azure C,    Basic fuchsine, Bismarck brown Y, Brilliant cresyl blue, Brilliant    green, Carmine, Chlorazol black E, Congo red, C.I. Cresyl violet,    Crystal violet, Darrow red, Eosin B, Eosin Y, Erythrosin, Ethyl    eosin, Ethyl green, Fast green F C F, Fluorescein Isothiocyanate,    Giemsa Stain, Hematoxylin, Hematoxylin & Eosin, Indigo carmine,    Janus green B, Jenner stain 1899, Light green SF, Malachite green,    Martius yellow, Methyl orange, Methyl violet 2B, Methylene blue,    Methylene blue, Methylene violet, (Bernthsen), Neutral red,    Nigrosin, Nile blue A, Nuclear fast red, Oil Red, Orange G, Orange    II, Orcein, Pararosaniline, Phloxin B, Protargol S, Pyronine B,    Pyronine, Resazurin, Rose Bengal, Safranine O, Sudan black B, Sudan    III, Sudan IV, Tetrachrome stain (MacNeal), Thionine, Toluidine    blue, Weigert, Wright stain, and any combination thereof.-   FB19. The method of any prior embodiment, wherein the detection    agent comprises antibodies configured to specifically bind to    protein analyte in the sample.-   FB20. The method of any prior embodiment, wherein the detection    agent comprises oligonucleotide probes configured to specifically    bind to DNA and/or RNA in the sample.-   FB21. The method of any prior embodiment, wherein the detection    agent is labeled with a reporter molecule, wherein the reporter    molecule is configured to provide a detectable signal to be read and    analyzed.-   FB22. The device of embodiment FB21, wherein the signal is selected    from the group consisting of:    -   i. luminescence selected from photoluminescence,        electroluminescence, and electrochemiluminescence;    -   ii. light absorption, reflection, transmission, diffraction,        scattering, or diffusion;    -   iii. surface Raman scattering;    -   iv. electrical impedance selected from resistance, capacitance,        and inductance;    -   v. magnetic relaxivity; and    -   vi. any combination of i-v.-   FB23. The method of any prior embodiment, wherein the sample contact    area of one or both of the plates comprise a binding site that    contains capture agents, wherein the capture agents are configured    to bind to the target analyte on the surface of cells in the sample    and immobilize the cells.-   FB24. The method of any prior embodiment, before step (e), further    comprising: incubating the sample at the closed configuration for a    period of time that is longer than the time it takes for the    detection agent to diffuse across the layer of uniform thickness and    the sample.-   FB25. The method of any prior embodiment, before step (e), further    comprising: incubating the sample at the closed configuration at a    predetermined temperature in the range of 30-75° C.-   FB26. The method of any prior embodiment, wherein the staining    liquid comprises the transfer solution.

G. Dual Lens Imaging System

But nowadays dual cameras are more and more common on state-of-artsmartphones, which offers more possibilities of smartphone basedimaging. By using two cameras, two different areas of the sample can beimaged at the same time, which is equivalent to a much larger field ofview. And what's more, each camera can be used to do microscopy imagingat a different resolution. For example, one camera can do microscopywith lower resolution but larger field of view to image large objects insample and the other camera can do microscopy with higher resolution butsmaller field of view to image small objects. This is useful when thesample for imaging has mixed small objects and large objects. Hence, itis highly desirable to provide the users the smartphone imaging systembased on dual cameras.

Dual Camera Imaging System

FIG. 19-A is the schematic illustration of the dual camera imagingsystem. The dual camera imaging system comprises a mobile computingdevice (e.g. smartphone) having two built-in camera modules, twoexternal lenses, a QMAX device and light source. Each camera module hasan internal lens and an image sensor. The QMAX device is located underthe two camera modules. Each external lens is placed between QMAX deviceand its corresponding internal lens at the appropriate height where thesample in QMAX device can be clearly focused on the image sensor. Eachexternal lens is aligned with its corresponding internal lens. The lightcaptured by the imaging sensor can be refracted from the specimen,emitted from the specimen, etc. The light captured by the imaging sensorcovers visible wavelength and can illuminate on the sample in QMAXdevice from back or top side in a normal or oblique incidence angle.

Dual Camera Imaging System for Large FOV Imaging

One embodiment is that the dual camera imaging system is used for largeFOV imaging. In this embodiment, the images taken by both camera havethe same scale or optical magnification. To achieve this, the focallength of external lens 1 f_(E1), the focal length of internal lens 1f_(N1), the focal length of external lens 2 f_(E2) and the focal lengthof internal lens 2 f_(N2) satisfy the relationship:

${\frac{fE1}{fN1} = \frac{fE2}{fN2}}.$

-   The distance between two cameras is chosen to an appropriate value    so that the FOVs of both cameras have overlap. As shown in FIG.    19-B, the letter “A” represents the sample, due to the overlap    between the FOVs of two cameras, some part of the letter “A” exist    in both the FOV of camera 1 and FOV of camera 2.-   A further image processing step is used to merge the two images into    one large image by matching the same feature shared by the two    images taken by camera 1 and camera 2.

Dual Camera Imaging System for Dual Resolution Imaging

The lens-based imaging system has the intrinsic drawback that it has thetrade-off between the size of FOV and resolution. To achieve large FOV,the resolution of the imaging system need to be sacrificed. This problemis more concerned when the sample has mixed small and large objects withsignificant different size scale. In order to image enough number oflarge objects the FOV need to be large enough, but that will lose theresolution to get the details of the small objects. To solve thisproblem, in this embodiment, the dual camera imaging system is used forachieve dual resolution imaging on a same sample, in which camera 1 (or2) is used for low resolution and large FOV imaging and camera 2 (or 1)is used for high resolution and small FOV imaging.

The resolution of the imaging system depends on the opticalmagnification and the optical magnification is equal to the ratio of thefocal length of the external lens to the focal length of the internallength. For example, in this embodiment, camera 1 is used for lowresolution imaging and camera 2 is used for high resolution imaging,then the focal length of external lens 1 f_(E1), the focal length ofinternal lens 1 f_(N1), the focal length of external lens 2 f_(E2) andthe focal length of internal lens 2 f_(N2) satisfy the relationship:

${\frac{fE1}{fN1} < \frac{fE2}{fN2}}.$

The FOVs of both cameras can have overlap or no overlap.

As shown in FIG. 19-C, the sample image taken by camera 1 covers largerFOV and contains more objects in a single FOV but cannot resolve thedetail of the small objects. And the image taken by camera 2 covers arelatively small FOV and contains fewer objects in a single FOV but hashigher resolution that can resolve the details in the small objects.

Examples of Present Invention

-   A1. A dual lens imaging device, comprising: a first external lens, a    second external lens, a housing unit, and a card unit, wherein: the    housing unit is configured to accommodate the first and second    external lenses and the card unit, and to connect the dual length    imaging device with a mobile device; the first and the second    external lenses are configured to respectively align with two    internal lenses in the mobile device; and the card unit is    configured to accommodate a specimen card, which contains a sample,    wherein the card unit is positioned between the external lenses and    the internal lenses; wherein the external lenses are configured to    focus illuminating light that is refracted or emitted from the    specimen card onto image sensors in the mobile device, allowing the    image sensors to capture images of the sample.-   B1. A dual lens imaging system, comprising: the dual lens imaging    device of embodiment A1, the mobile device, which comprises hardware    and software to capture and process images of the sample through the    dual lens imaging device.-   C1. The device or system of any prior embodiments, wherein the    specimen card is a QMAX card.-   C2. The device or system of any prior embodiments, wherein the    mobile device is a mobile communication device.-   C3. The device or system of any prior embodiments, wherein mobile    device is a smart phone.-   C4. The device or system of any prior embodiments, wherein the    mobile device comprises a light source, which provides light to the    specimen card.-   C5. The device or system of any prior embodiments, wherein the two    external lenses are configured to capture overlapping images that    are at least partly overlapping.-   C6. The device or system or embodiment C5, wherein the overlapping    images have the same resolution.-   C7. The device or system of embodiment C6, wherein the software is    configured to process the overlapping images to generate a combined    image of the sample.-   C8. The device or system of embodiment C5, wherein the overlapping    images have the different resolutions.-   C9. The device or system of embodiment C8, wherein the software is    configured to process the overlapping images illustrate specific    portions of the image having lower resolution.-   C10. The device or system of any prior embodiment, wherein the two    external lenses are configured to image two different locations of a    sample area of the Q-Card.-   C11. The device or system of any prior embodiment, wherein the two    external lenses are configured to have different size of FoV (field    of view from each other).-   C12. The device or system of any prior embodiment, wherein the two    external lenses are configured to have different size of FoV (field    of view from each other), and wherein the ratio of the two different    of FoV is 1.1, 1.2, 1.5, 2, 5, 10, 15, 20, 30, 50, 100, 200, 1000,    or in a range of any value of the two. A preferred ratio is 1.2,    1.5, 2, 5, 10, 20, or in a range of any value of the two.-   C13. The device or system of any prior embodiment, wherein overlap    of FoV of the two external lenses are configured to be around 1%,    5%, 10%, 20%, 50%, 60%, 70%, 80%, 90%, or a range between any two of    these values.-   C14. The device or system of any prior embodiment, wherein the two    external lenses are optically coupled with different optical filters    and/or polarizers.

Other Embodiments

An optical adaptor for imaging an sample using a hand-held imagingdevice that has a light source, a single camera, and a computerprocessor, comprising: an enclosure; a cavity within the enclosure; anda lever within the cavity, wherein the lever comprises at least oneoptical element and is configured to be moveable between a firstposition and a second position, wherein (i) in the first position, saidimaging device is capable of imaging a sample in a bright field mode,and (ii) in the second position, said imaging device is capable ofimaging the sample in a fluorescence excitation mode.

An optical adaptor for imaging an sample using a hand-held imagingdevice that has a light source, a single camera, and a computerprocessor, comprising: an enclosure; a lens arranged to provide a fieldof view for the camera; a cavity within the enclosure for receiving thesample and positioning the sample within the field of view of thecamera, wherein the lens is positioned to receive light refracted by oremitted by the sample when in the field of view of the camera; and alever within the cavity, wherein the lever comprises at least oneoptical element and is configured to be moveable between a firstposition and a second position, wherein (i) in the first position, saidimaging device is capable of imaging a sample in a bright field mode,and (ii) in the second position, said imaging device is capable ofimaging the sample in a fluorescence excitation mode.

An optical adaptor for imaging an sample using a hand-held imagingdevice that has a light source, a single camera, and a computerprocessor, comprising: an enclosure; a cavity within the enclosure forreceiving the sample and positioning the sample within a field of viewof the camera; and a lever within the cavity, wherein the levercomprises at least one optical element and is configured to be moveablebetween a first position and a second position, wherein (i) in the firstposition, said imaging device is capable of imaging a sample in a brightfield mode, and (ii) in the second position, said imaging device iscapable of imaging the sample in a fluorescence excitation mode, andwherein the lever comprises a first planar region extending along afirst plane and a second planar region laterally displaced along a firstdirection from the first planar region and extending along a secondplane, the first plane being disposed at a different height along asecond direction from the second plane, the second direction beingorthogonal to the first direction.

An optical adaptor for imaging an sample using a hand-held imagingdevice that has a light source, a single camera, and a computerprocessor, comprising: an enclosure; a cavity within the enclosure forreceiving the sample and positioning the sample within a field of viewof the camera; and a lever within the cavity, wherein the levercomprises at least one optical element and is configured to be moveablebetween a first position and a second position, wherein (i) in the firstposition, said imaging device is capable of imaging a sample in a brightfield mode, and (ii) in the second position, said imaging device iscapable of imaging the sample in a fluorescence excitation mode, andwherein the lever comprises a first planar region extending along afirst plane and a second planar region laterally displaced along a firstdirection from the first planar region and extending along a secondplane, the first plane being disposed at a different height along asecond direction from the second plane, the second direction beingorthogonal to the first direction, and wherein the first planar regioncomprises at least one optical element, and the second planar regioncomprises at least one optical element.

An optical adaptor for imaging an sample using a hand-held imagingdevice that has a light source, a single camera, and a computerprocessor, comprising: an enclosure; a cavity within the enclosure; anda lever within the cavity, wherein the lever comprises at least oneoptical element and is configured to be moveable between at least threedifferent positions, wherein (i) in a first position, said imagingdevice is capable of imaging a sample in a bright field mode, (ii) in asecond position, said imaging device is capable of imaging the sample ina fluorescence excitation mode, and (iii) in a third position, saidimaging device is capable of measuring optical absorption of the sample.

An optical adaptor for imaging an sample using a hand-held imagingdevice that has a light source, a single camera, and a computerprocessor, comprising: an enclosure; a lens configured to provide afield of view for the camera; a cavity within the enclosure forreceiving the sample and positioning the sample within the field of viewof the camera; an aperture within the enclosure, wherein the aperture isarranged to receive source light from the light source for illuminatingthe sample; and a lever within the cavity, wherein the lever comprisesat least one optical element and is configured to be moveable between afirst position and a second position, wherein (i) in a first position,said imaging device is capable of imaging a sample in a bright fieldmode, (ii) in a second position, said imaging device is capable ofimaging the sample in a fluorescence excitation mode, wherein in thefluorescence excitation mode, the lens is arranged to receive lightemitted by the sample when the sample is illuminated by the sourcelight.

An optical adaptor for imaging an sample using a smart phone that has alight source, a single camera, and a computer processor, comprising: anenclosure; a lens configured to provide a field of view for the camera;a cavity within the enclosure for receiving the sample and positioningthe sample within the field of view of the camera; and a lever withinthe cavity, wherein the lever comprises at least one optical element andis configured to be moveable between a first position and a secondposition, wherein (i) in a first position, said imaging device iscapable of imaging a sample in a bright field mode, and (ii) in a secondposition, said imaging device is capable of imaging the sample in afluorescence excitation mode.

An optical assembly attachable to a hand-held electronic device having alight source, a camera, and a computer processor, wherein the opticalassembly is configured to enable microscopic imaging of a sample by thecamera with illumination of the sample by light from the light source,the optical assembly comprising: an enclosure; a cavity within theenclosure; a lens configured to provide a microscopic field of view forthe camera; and moveable arm within the cavity, wherein the moveable armis configurable to switch between a first position and a secondposition, wherein when the moveable arm is in the first position, theoptical assembly is in a bright field mode, and when the moveable arm isin the second position, the optical assembly is in a fluorescenceexcitation mode.

The optical assembly of any embodiments, wherein the enclosurecomprises: a sample receptacle region within the cavity; and a slot on aside of the enclosure, wherein the slot is arranged to receive a samplesubstrate within the sample receptacle region and position the samplewithin the field of view of the camera.

The optical assembly of embodiments, further comprising a first set ofone or more optical elements arranged to receive light entering from afirst aperture in the enclosure corresponding to the light source and toredirect the light entering from the first aperture along a firstpathway toward a second aperture in the enclosure corresponding to thecamera to provide bright field illumination of the sample when themoveable arm is in the first position.

The optical assembly of embodiments, wherein the first set of one ormore optical elements comprises a first right angle mirror and a secondright angle mirror, wherein the first right angle mirror and the secondright angle mirror are in the first pathway and are arranged to reflectthe light from the light source to be normally incident into the camera,

The optical assembly of embodiments, wherein the light source is a pointsource to achieve interference imaging of transparent samples viailluminating the sample by a same wavefront.

The optical assembly of embodiments, further comprising a second set ofone or more optical elements mechanically coupled to the movable arm andarranged to receive light entering from the first aperture and redirectthe light entering from the first aperture along a second pathway toobliquely illuminate the sample to provide fluorescence illumination ofthe sample when the moveable arm is in the second position,

The optical assembly of embodiments, wherein the oblique angle is largerthan a collecting angle of the lens configured to provide the field ofview of the camera.

The optical assembly of embodiments, wherein the second set of one ormore optical elements includes a mirror and an optical absorber, whereinthe mirror reflects light to obliquely illuminate the sample and theoptical absorber absorbs extraneous light from the first aperture thatwould otherwise pass through the second aperture of the enclosure andoverwhelm the camera in the fluorescence excitation mode.

The optical assembly of embodiments, wherein the absorber absorbs lightthat is not incident on the mirror after going through the firstaperture, wherein the light absorber is a thin-film light absorber.

The optical assembly of embodiments, further comprising a third set ofone or more optical elements arranged to receive light entering from thefirst aperture and redirect the light entering into the second aperturein the movable arm and going along the first pathway toward a lightdiffuser on the movable arm to illuminate the sample in normal directionto measure the optical absorption of the sample.

The optical assembly of embodiments, wherein the third set of one ormore optical elements includes a light diffuser, a first right anglemirror and a second right angle mirror, wherein the first right anglemirror and the second right angle mirror are in the first pathway andare arranged to reflect the light from the light source toward the lightdiffuser and then to be normally incident into the camera;

The optical assembly of embodiments, wherein the light diffuser is asemi-opaque diffuser with opacity in the range of 10% to 90%.

The optical assembly of embodiments, further comprising a rubber door tocover the sample receptacle to prevent ambient light from entering intothe cavity.

The optical assembly of any of the preceding any embodiments, whereinthe light source and the camera are positioned on the same side of thehand-held electronic device at a fixed distance to one another.

A system comprising: the optical assembly of any of the preceding anyembodiments, and a mobile phone attachment comprising a first sideconfigured to couple to the optical assembly and a second opposite sideconfigured to couple to the hand-held electronic device, wherein thehand-held electronic device is a mobile phone.

The system of any embodiments, wherein the mobile phone attachment isexchangeable to provide attachment to different sized mobile phones.

The system of any embodiments, wherein a size of the mobile phoneattachment is adjustable.

An optical assembly for a hand-held mobile electronic device, theoptical assembly comprising: an enclosure; a cavity within theenclosure; a plurality of optical elements within the cavity, whereinthe plurality of optical elements are arranged to receive light enteringfrom a first aperture in the enclosure and to redirect the lightentering from the first aperture along a first pathway toward a secondaperture in the enclosure; a moveable arm configurable in at least twodifferent positions within the enclosure, a moveable arm configurable inat least three different positions within the enclosure, wherein themoveable arm comprises a light reflector portion to reflect light,wherein the moveable arm comprise a light diffuser to homogenize thelight and break the coherence of the light, wherein the moveable armcomprise an aperture aligned with the entrance aperture in theenclosure, wherein, when the moveable arm is in a first position withinthe enclosure, the light reflector portion is positioned between anentrance aperture in the enclosure and the plurality of optical elementssuch that the light reflector portion blocks the light entering from thefirst opening from being incident on the plurality of optical elements,and wherein, when the moveable arm is in a second position within theenclosure, the light entering from the first opening is incident on theplurality of optical elements, and wherein when the moveable arm is in athird position within the enclosure, the light entering from the firstopening goes through an aperture on the moveable arm and then isincident on the light diffuser;

The optical assembly of any embodiments, comprising a slot on a side ofthe enclosure, wherein the slot is arranged to receive a samplesubstrate such that: when the sample substrate is fully inserted withinthe slot and the moveable arm is in the second position within theenclosure, the first pathway intersects the sample substrate; and whenthe sample substrate is fully inserted within the slot and moveable armis in the first position within the enclosure, light reflected by thelight reflector portion is redirected to the sample substrate; and whenthe sample substrate is fully inserted within the slot and moveable armis the third position within the enclosure, light goes along the firstpathway toward a light diffuser and then illuminate on the samplesubstrate.

The optical assembly of any embodiments, wherein the moveable armcomprises a light absorber portion to absorb light that is not incidenton the mirror after going through the first aperture.

The optical assembly of any embodiments, wherein the moveable armcomprises: a first receptacle positioned above the light reflectorportion; and an optical filter seated in the receptacle; and a secondreceptacle positioned above the aperture portion; and a optical filterseated in the receptacle.

The optical assembly of any embodiments, wherein, when the moveable armis in the first position, the optical filter seated in the receptacle ispositioned to receive light entering from the first aperture in theenclosure; and when the moveable arm is in the third position, theoptical filter seated in the receptacle is positioned to receive lightentering from the first aperture in the enclosure.

The optical assembly of any embodiments, wherein, when the moveable armis in the first position, the optical filter seated in the receptacleoverlaps a region in which a portion of the sample substrate is locatedwhen the sample substrate is fully inserted within the slot.

A system comprising: the optical assembly of any embodiments; and amobile phone attachment comprising a first side configured to couple tothe optical assembly and comprising a second opposite side configured tocouple to a mobile phone, wherein a size of the mobile phone attachmentis adjustable.

An optical assembly attachable to a hand-held electronic device having alight source, a camera, and a computer processor, wherein the opticalassembly is configured to enable microscopic imaging of a sample by thecamera with illumination of the sample by light from the light source,the optical assembly comprising: a lens configured to provide amicroscopic field of view for the camera; a receptacle for receiving thesample and positioning the sample within the microscopic field of view;and an optical fiber configured to receive the light from the lightsource and to illuminate the receptacle.

The optical assembly of any embodiments wherein, when the opticalassembly is attached to the hand-held electronic device, the lens andthe camera define an optical axis, and wherein the optical fibercircumscribes the optical axis.

The optical assembly of any embodiments wherein the optical fiber isring-shaped.

The optical assembly of any embodiments wherein the optical fiber is aside-emitting fiber.

The optical assembly of any embodiments wherein the optical assemblycomprises an enclosure defining the receptacle, wherein the ring-shapedfiber sits in a groove of the enclosure, wherein the enclosure comprisesan aperture configured to align with the light source and both end facesof the ring-shape fiber to receive light from the light source.

The optical assembly of any embodiments, wherein the light emits fromthe side of the ring-shape fiber to illuminate the sample area rightunder the camera in the optical axis.

The optical assembly of any embodiments, wherein the optical assemblycomprises an enclosure defining the receptacle, wherein the enclosurecomprises a first aperture configured to align with the light source,and a first end face of the optical fiber is positioned in the firstaperture to receive light from the light source.

The optical assembly of any embodiments wherein the enclosure comprisesa second aperture configured to align with the camera, and wherein theoptical fiber comprises a first end positioned in the first aperture andcomprises a second end positioned in the second aperture.

The optical assembly of any embodiments wherein at least one of thefirst end face of the optical fiber and a second end face of the opticalfiber is matted.

The optical assembly of any embodiments wherein when the opticalassembly is attached to the hand-held electronic device, the opticalfiber is tilted with respect to the light source, and wherein a secondend face of the optical fiber is arranged to illuminate a region of thesample located directly beneath the lens.

The optical assembly of any embodiments wherein the optical assemblycomprises an enclosure defining the receptacle, the enclosure comprisesa groove, and the optical fiber is arranged in the groove.

An optical assembly attachable to a hand-held electronic device having alight source, a camera, and a computer processor, wherein the opticalassembly is configured to enable microscopic fluorescence imaging of asample by the camera with illumination of the sample by light from thelight source, the optical assembly comprising: a lens configured toprovide a microscopic field of view for the camera; a receptacle forreceiving the sample and positioning the sample within the microscopicfield of view; a mirror off-set from an optical axis of the lens andpositioned to reflect light from the light source and illuminate thesample over a range of oblique angles with respect to the optical axis;and a wavelength filter positioned between the sample and the camera topass fluorescence emitted by the sample in response to the obliqueillumination.

The optical assembly of any embodiments wherein the lens is positionedon a front-side of the sample and the mirror is positioned to obliquelyilluminate the sample from a back-side of the sample, wherein theoblique angle is larger than a collecting angle of the lens.

The optical assembly of any embodiments further comprising an opticalabsorber positioned on the optical axis adjacent the mirror to absorblight from the light source not reflected by the mirror.

The optical assembly of any embodiments wherein the mirror and theoptical absorber are mounted on a common structure and tilted withrespect to one another.

The optical assembly of any embodiments, further comprising a secondwavelength filter positioned in a path of the illumination light betweenthe light source and the mirror to select certain wavelengths forilluminating the sample.

The optical assembly of any of the preceding any embodiments, whereinthe sample is supported by a sample holder comprising a planarstructure, and wherein the receptacle is configured to position theplanar structure to extend partially into a path of illumination lightfrom the light source to couple illumination light into the planarstructure.

The optical assembly of any embodiments 6, wherein the receptacle isconfigured to position the planar structure such that the path ofillumination light is incident on an edge of the planar structure,wherein the edge extends along a plane that is normal to a planecomprising the field of view.

The optical assembly of any embodiments wherein the mirror is arrangedto reflect the light to partially obliquely illuminate the sample from aback side of the planar structure and to partially illuminate an edge ofthe planar structure to couple illumination light into the planarstructure.

The optical assembly of any embodiments further comprising a rubber doorto cover the sample receptacle to prevent ambient light from enteringthe optical assembly and entering the camera.

The optical assembly of any embodiments, wherein the planar structure isconfigured to waveguide the coupled illumination light to the sample toilluminate the sample and cause the sample to emit fluorescence.

The optical assembly of any embodiments further comprising the sampleholder,

The optical assembly of any embodiments 6, wherein the sample is aliquid sample and the sample holder comprises first and second platessandwiching the liquid sample.

The optical assembly of any of the preceding any embodiments, whereinthe lens, the receptacle, the mirror, and the wavelength filter aresupported in a common optical box and further comprising an exchangeableholder frame for attaching the optical box to the hand-held electronicdevice.

The optical assembly of any embodiments, wherein the light source andthe camera are positioned on the same side of the hand-held electronicdevice and at fixed distance to one another.

The optical assembly of any embodiments, wherein the hand-heldelectronic device is a smart phone.

An apparatus comprising the optical assembly of any of the preceding anyembodiments and the hand-held electronic device.

An optical assembly attachable to a hand-held electronic device having alight source, a camera, and a computer processor, the optical assemblyconfigured to enable microscopic fluorescence imaging of a sample by thecamera with illumination of the sample by light from the light source,the optical assembly comprising: a lens configured to provide amicroscopic field of view for the camera; a receptacle for receiving thesample and positioning the sample within the microscopic field of view,wherein the sample is supported by a sample holder comprising a planarstructure, and wherein the receptacle is configured to position theplanar structure to extend partially into a path of illumination lightfrom the light source to couple illumination light into the planarstructure and cause the sample to emit fluorescence; and a wavelengthfilter positioned between the sample and the camera to pass fluorescenceemitted by the sample in response to the illumination.

The optical assembly of any embodiments further comprising a rubber doorto cover the sample receptacle to prevent ambient light entering theoptical assembly through the receptacle.

The optical assembly of any embodiments wherein the planar structure isconfigured to waveguide the coupled illumination light to the sample toilluminate the sample and cause the sample to emit the fluorescence.

The optical assembly of any embodiments further comprising the sampleholder,

The optical assembly of any embodiments wherein the sample is a liquidsample and the sample holder comprises first and second platessandwiching the liquid sample.

The optical assembly of any embodiments, further comprising a secondwavelength filter positioned in the path of the illumination lightbetween the light source and the portion of the sample holder partiallyextending into the path of the light.

The optical assembly of any of the preceding any embodiments, whereinthe lens, the receptacle, and the wavelength filter are supported in acommon optical box and further comprising an exchangeable holder framefor attaching the optical box to the hand-held electronic device.

The optical assembly of any embodiments wherein the light source and thecamera are positioned on the same side of the hand-held electronicdevice at a fixed distance to one another.

The optical assembly of any embodiments, wherein the hand-heldelectronic device is a smart phone.

An apparatus comprising the optical assembly of any of the preceding anyembodiments and the hand-held electronic device.

An optical assembly attachable to a hand-held electronic device having alight source, a first camera module, a second camera module, and acomputer processor, wherein the optical assembly is configured to enablemicroscopic imaging of a sample by the first camera and by the secondcamera with illumination of the sample by light from the light source,the optical assembly comprising: a first assembly lens configured toprovide a first microscopic field of view for the first camera module; asecond assembly lens configured to provide a second microscopic field ofview for the second camera module; and a receptacle for receiving thesample and positioning the sample within the first microscopic field ofview and within the second microscopic field of view.

The optical assembly of any embodiments, wherein the first camera modulecomprises a first internal lens and the second camera module comprises asecond internal lens, wherein a first optical magnification provided bythe first assembly lens and the first internal lens is the same as asecond optical magnification provided by the second assembly lens andthe second internal lens.

The optical assembly of any embodiments, wherein a first ratio of afocal length of the first assembly lens to a focal length of the firstinternal lens is equal to a second ratio of a focal length of the secondassembly lens to a focal length of the second internal lens.

The optical assembly of any embodiments, wherein a first imageresolution provided by the first camera module and the first assemblylens is the same as a second image resolution provided by the secondcamera module and the second assembly lens.

The optical assembly of any embodiments, wherein the first camera modulecomprises a first internal lens and the second camera module comprises asecond internal lens, wherein a first optical magnification provided bythe first assembly lens and the first internal lens is different from asecond optical magnification provided by the second assembly lens andthe second internal lens.

The optical assembly of any embodiments, wherein a first ratio of afocal length of the first assembly lens to a focal length of the firstinternal lens is less than a second ratio of a focal length of thesecond assembly lens to a focal length of the second internal lens.

The optical assembly of any embodiments, wherein a first imageresolution provided by the first camera module and the first assemblylens is less than a second image resolution provided by the secondcamera module and the second assembly lens.

The optical assembly of any of the preceding any embodiments, whereinthe first microscopic field of view overlaps with the second microscopicfield of view.

The optical assembly of any embodiments, wherein an amount of overlap ofthe first microscopic field of view with the second microscopic field ofview is between 1% and 90%.

The optical assembly of any of any embodiments, wherein the firstmicroscopic field of view does not overlap with the second microscopicfield of view.

The optical assembly of any of the preceding any embodiments, whereineach of the first assembly lens and the second assembly lens is arrangedto receive light scattered by or emitted by the sample.

The optical assembly of any of the preceding any embodiments, whereinthe first microscopic field of view is less than the second microscopicfield of view.

The optical assembly of any of the preceding any embodiments, wherein anangular field of view of the first assembly lens is less than an angularfield of view of the second assembly lens.

The optical assembly of any embodiments, wherein a ratio of the angularfield of view of the first assembly lens to the angular field of thesecond assembly lens is between 1.1 and 1000.

The optical assembly of any of the preceding any embodiments,comprising: a first optical filter arranged in a first illumination pathto or from the first assembly lens; and a second optical filter arrangedin a second illumination path to or from the second assembly lens.

The optical assembly of any embodiments, wherein the first opticalfilter is configured to filter a first range of wavelengths, the secondoptical filter is configured to filter a second range of wavelengths,and the first range of wavelengths is different from the second range ofwavelengths.

The optical assembly of any of the preceding any embodiments,comprising: a first polarizer arranged in a first illumination path toor from the first assembly lens; and a second polarizer arranged in asecond illumination path to or from the second assembly lens.

The optical assembly of any embodiments, wherein the first polarizer andthe second polarizer have different polarization dependent lighttransmission and blocking properties.

An apparatus comprising the optical assembly of any of the preceding anyembodiments and the hand-held electronic device.

The apparatus of any embodiments, wherein the hand-held electronicdevice is a smart phone.

The apparatus of any embodiments, wherein the hand-held electronicdevice is configured to computationally merge a first image obtainedfrom the first camera module with a second image obtained from thesecond camera module.

An imaging method comprising: compressing a sample between two plates,wherein the two plates are separated from one another by an array ofspacers, at least one of which has a reference mark; acquiring multipleimages of the sample using an imaging system comprising a camera and atleast one lens, wherein each image corresponds to a different objectplane within a thickness of the sample; computationally analyzing eachimage to determine information about the corresponding object planebased on one or more of the reference marks; and computationallyconstructing a three-dimensional image of the sample based on themultiple images and the information about the corresponding objectplanes.

The imaging method of any embodiments, wherein the determinedinformation about the corresponding object plane comprises a depth ofthe object plane relative to imaging system.

The imaging method of any embodiments of any embodiments 2, wherein atleast some of the spacers each have a reference mark.

The imaging method of any embodiments, wherein the determinedinformation about the corresponding object plane comprises a depth andan orientation of the object plane relative to imaging system.

The imaging method of any of the preceding any embodiments, where thecomputational analyzing of each image comprises determining a degree ofdefocus of one or more of the reference marks.

The imaging method of any embodiments, where the computational analyzingof each image comprises determining a depth for each of multiple ones ofthe reference marks based on a degree of defocus for each such referencemark and determining a depth and an orientation of the correspondingobject plane relative to the imaging system based on the determineddepths of the reference marks.

The imaging method of any of the preceding any embodiments, wherein thereferences marks are not rotationally symmetric with respect to an axisperpendicular to at least one of the plates.

The imaging method of any embodiments, wherein the computationalanalyzing of each image comprises determining a rotational orientationof one or more of the reference marks about the axis relative to theimaging system.

The imaging method of any of the preceding any embodiments, wherein thecomputational analyzing of each image comprising comparing imageinformation about the reference marks to a priori knowledge about thereference marks.

The imaging method of any embodiments, wherein the a priori knowledgeabout the reference marks is based on one or more of a shape of eachreference mark and a location of each reference mark relative to theplates.

The imaging method of any of the preceding any embodiments, wherein thespacers are pillars.

The imaging method of any of the preceding any embodiments, wherein theacquiring of the multiple images comprises moving one or more componentsof the imaging system relative to the plates sandwiching the sample.

The imaging method of any of the preceding any embodiments, wherein thecomputational constructing of the three-dimensional image comprisesprocessing each acquired image to remove out-of-focus features.

The imaging method of any embodiments, wherein the processing of eachacquired image to remove out-of-focus features comprises using aband-pass filter.

The imaging method of any of the preceding any embodiments, where theacquired images correspond to interference images formed by combininglight from the sample with reference light not directed to the sample onthe camera.

An imaging apparatus comprising: an imaging system comprising a cameraand at least one lens; a sample holder for supporting a sample cartridgerelative to the imaging system, the sample cartridge comprising twoplates are separated from one another by an array of spacers, at leastone of which has a reference mark, wherein a sample to be imaged isconfigured to be compressed between the two plates; and a processing andcontrol system coupled to the sample holder and the camera andconfigured to acquire multiple images of the sample using the imagingsystem, wherein each image corresponds to a different object planewithin a thickness of the sample, and wherein the processing and controlsystem is further configured to: computationally analyze each image todetermine information about the corresponding object plane based on oneor more of the reference marks; and computationally construct athree-dimensional image of the sample based on the multiple images andthe information about the corresponding object planes.

The imaging apparatus of any embodiments, wherein the determinedinformation about the corresponding object plane comprises a depth ofthe object plane relative to imaging system.

The imaging apparatus of any embodiments or, wherein at least some ofthe spacers each have a reference mark.

The imaging apparatus of any embodiments, wherein the determinedinformation about the corresponding object plane comprises a depth andan orientation of the object plane relative to imaging system.

The apparatus of any of the preceding any embodiments, where thecomputational analyzing of each image comprises determining a degree ofdefocus of one or more of the reference marks.

The apparatus of any embodiments 20, wherein the computational analyzingof each image comprises determining a depth for each of multiple ones ofthe reference marks based on a degree of defocus for each such referencemark and determining a depth and an orientation of the correspondingobject plane relative to the imaging system based on the determineddepths of the reference marks.

The apparatus of any of the preceding any embodiments, wherein thereferences marks are not rotationally symmetric with respect to an axisperpendicular to at least one of the plates.

The apparatus of any embodiments, wherein the computational analyzing ofeach image comprises determining a rotational orientation of one or moreof the reference marks about the axis relative to the imaging system.

The apparatus of any of the preceding any embodiments, wherein thecomputational analyzing of each image comprising comparing imageinformation about the reference marks to a priori knowledge about thereference marks.

The apparatus of any embodiments, wherein the a priori knowledge aboutthe reference marks is based on one or more of a shape of each referencemark and a location of each reference mark relative to the plates.

The apparatus of any of the preceding any embodiments, wherein thespacers are pillars.

The apparatus of any of the preceding any embodiments, wherein thecontrol system is configured to move one or more components of theimaging system relative to the plates sandwiching the sample to acquirethe multiple images.

The apparatus of any of the preceding any embodiments, wherein thecomputational constructing of the three-dimensional image comprisesprocessing each acquired image to remove out-of-focus features.

The apparatus of any embodiments wherein the processing of each acquiredimage to remove out-of-focus features comprises using a band-passfilter.

The apparatus of any of the preceding any embodiments, wherein where theacquired images correspond to interference images formed by combininglight from the sample with reference light not directed to the sample onthe camera.

More Other Embodiments

The present invention includes a variety of embodiments, which can becombined in multiple ways as long as the various components do notcontradict one another. The embodiments should be regarded as a singleinvention file: each filing has other filing as the references and isalso referenced in its entirety and for all purpose, rather than as adiscrete independent. These embodiments include not only the disclosuresin the current file, but also the documents that are herein referenced,incorporated, or to which priority is claimed.

(1) Definitions

The terms used in describing the devices, systems, and methods hereindisclosed are defined in the current application, or in PCT Application(designating U.S.) Nos. PCT/US2016/045437 and PCT/US0216/051775, whichwere respectively filed on Aug. 10, 2016 and Sep. 14, 2016, U.S.Provisional Application No. 62/456,065, which was filed on Feb. 7, 2017,U.S. Provisional Application No. 62/426,065, which was filed on Feb. 8,2017, U.S. Provisional Application No. 62/456,504, which was filed onFeb. 8, 2017, all of which applications are incorporated herein in theirentireties for all purposes.

The terms “CROF Card (or card)”, “COF Card”, “QMAX-Card”, “Q-Card”,“CROF device”, “COF device”, “QMAX-device”, “CROF plates”, “COF plates”,and “QMAX-plates” are interchangeable, except that in some embodiments,the COF card does not comprise spacers; and the terms refer to a devicethat comprises a first plate and a second plate that are movablerelative to each other into different configurations (including an openconfiguration and a closed configuration), and that comprises spacers(except some embodiments of the COF card) that regulate the spacingbetween the plates. The term “X-plate” refers to one of the two platesin a CROF card, wherein the spacers are fixed to this plate. Moredescriptions of the COF Card, CROF Card, and X-plate are given in theprovisional application Ser. No. 62/456,065, filed on Feb. 7, 2017,which is incorporated herein in its entirety for all purposes.

(2) Q-Card, Spacer and Uniform Sample Thickness

The devices, systems, and methods herein disclosed can include or useQ-cards, spacers, and uniform sample thickness embodiments for sampledetection, analysis, and quantification. In some embodiments, the Q-cardcomprises spacers, which help to render at least part of the sample intoa layer of high uniformity. The structure, material, function, variationand dimension of the spacers, as well as the uniformity of the spacersand the sample layer, are herein disclosed, or listed, described, andsummarized in PCT Application (designating U.S.) Nos. PCT/US2016/045437and PCT/US0216/051775, which were respectively filed on Aug. 10, 2016and Sep. 14, 2016, U.S. Provisional Application No. 62/456,065, whichwas filed on Feb. 7, 2017, U.S. Provisional Application No. 62/426,065,which was filed on Feb. 8, 2017, U.S. Provisional Application No.62/456,504, which was filed on Feb. 8, 2017, all of which applicationsare incorporated herein in their entireties for all purposes.

(3) Hinges, Opening Notches, Recessed Edge and Sliders

The devices, systems, and methods herein disclosed can include or useQ-cards for sample detection, analysis, and quantification. In someembodiments, the Q-card comprises hinges, notches, recesses, andsliders, which help to facilitate the manipulation of the Q card and themeasurement of the samples. The structure, material, function, variationand dimension of the hinges, notches, recesses, and sliders are hereindisclosed, or listed, described, and summarized in PCT Application(designating U.S.) Nos. PCT/US2016/045437 and PCT/US0216/051775, whichwere respectively filed on Aug. 10, 2016 and Sep. 14, 2016, U.S.Provisional Application No. 62/456,065, which was filed on Feb. 7, 2017,U.S. Provisional Application No. 62/426,065, which was filed on Feb. 8,2017, U.S. Provisional Application No. 62/456,504, which was filed onFeb. 8, 2017, all of which applications are incorporated herein in theirentireties for all purposes.

(4) Q-Card, Sliders, and Smartphone Detection System

The devices, systems, and methods herein disclosed can include or useQ-cards for sample detection, analysis, and quantification. In someembodiments, the Q-cards are used together with sliders that allow thecard to be read by a smartphone detection system. The structure,material, function, variation, dimension and connection of the Q-card,the sliders, and the smartphone detection system are herein disclosed,or listed, described, and summarized in PCT Application (designatingU.S.) Nos. PCT/US2016/045437 and PCT/US0216/051775, which wererespectively filed on Aug. 10, 2016 and Sep. 14, 2016, U.S. ProvisionalApplication No. 62/456,065, which was filed on Feb. 7, 2017, U.S.Provisional Application No. 62/426,065, which was filed on Feb. 8, 2017,U.S. Provisional Application No. 62/456,504, which was filed on Feb. 8,2017, all of which applications are incorporated herein in theirentireties for all purposes.

In some embodiments of QMAX, the sample contact area of one or both ofthe plates comprises a compressed open flow monitoring surfacestructures (MSS) that are configured to monitoring how much flow hasoccurred after COF. For examples, the MSS comprises, in someembodiments, shallow square array, which will cause friction to thecomponents (e.g. blood cells in a blood) in a sample. By checking thedistributions of some components of a sample, one can obtain informationrelated to a flow, under a COF, of the sample and its components.

The depth of the MSS can be 1/1000, 1/100, 1/100, ⅕, ½ of the spacerheight or in a range of any two values, and in either protrusion or wellform.

(5) Detection Methods

The devices, systems, and methods herein disclosed can include or beused in various types of detection methods. The detection methods areherein disclosed, or listed, described, and summarized in PCTApplication (designating U.S.) Nos. PCT/US2016/045437 andPCT/US0216/051775, which were respectively filed on Aug. 10, 2016 andSep. 14, 2016, U.S. Provisional Application No. 62/456,065, which wasfiled on Feb. 7, 2017, U.S. Provisional Application No. 62/426,065,which was filed on Feb. 8, 2017, U.S. Provisional Application No.62/456,504, which was filed on Feb. 8, 2017, all of which applicationsare incorporated herein in their entireties for all purposes.

(6) Labels

The devices, systems, and methods herein disclosed can employ varioustypes of labels that are used for analytes detection. The labels areherein disclosed, or listed, described, and summarized in PCTApplication (designating U.S.) Nos. PCT/US2016/045437 andPCT/US0216/051775, which were respectively filed on Aug. 10, 2016 andSep. 14, 2016, U.S. Provisional Application No. 62/456,065, which wasfiled on Feb. 7, 2017, U.S. Provisional Application No. 62/426,065,which was filed on Feb. 8, 2017, U.S. Provisional Application No.62/456,504, which was filed on Feb. 8, 2017, all of which applicationsare incorporated herein in their entireties for all purposes.

(7) Analytes

The devices, systems, and methods herein disclosed can be applied tomanipulation and detection of various types of analytes (includingbiomarkers). The analytes and are herein disclosed, or listed,described, and summarized in PCT Application (designating U.S.) Nos.PCT/US2016/045437 and PCT/US0216/051775, which were respectively filedon Aug. 10, 2016 and Sep. 14, 2016, U.S. Provisional Application No.62/456,065, which was filed on Feb. 7, 2017, U.S. ProvisionalApplication No. 62/426,065, which was filed on Feb. 8, 2017, U.S.Provisional Application No. 62/456,504, which was filed on Feb. 8, 2017,all of which applications are incorporated herein in their entiretiesfor all purposes.

(8) Applications (Field and Samples)

The devices, systems, and methods herein disclosed can be used forvarious applications (fields and samples). The applications are hereindisclosed, or listed, described, and summarized in PCT Application(designating U.S.) Nos. PCT/US2016/045437 and PCT/US0216/051775, whichwere respectively filed on Aug. 10, 2016 and Sep. 14, 2016, U.S.Provisional Application No. 62/456,065, which was filed on Feb. 7, 2017,U.S. Provisional Application No. 62/426,065, which was filed on Feb. 8,2017, U.S. Provisional Application No. 62/456,504, which was filed onFeb. 8, 2017, all of which applications are incorporated herein in theirentireties for all purposes.

(9) Cloud

The devices, systems, and methods herein disclosed can employ cloudtechnology for data transfer, storage, and/or analysis. The relatedcloud technologies are herein disclosed, or listed, described, andsummarized in PCT Application (designating U.S.) Nos. PCT/US2016/045437and PCT/US0216/051775, which were respectively filed on Aug. 10, 2016and Sep. 14, 2016, U.S. Provisional Application No. 62/456,065, whichwas filed on Feb. 7, 2017, U.S. Provisional Application No. 62/426,065,which was filed on Feb. 8, 2017, U.S. Provisional Application No.62/456,504, which was filed on Feb. 8, 2017, all of which applicationsare incorporated herein in their entireties for all purposes.

Additional Notes

Further examples of inventive subject matter according to the presentdisclosure are described in the following enumerated paragraphs.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise, e.g., when the word “single” isused. For example, reference to “an analyte” includes a single analyteand multiple analytes, reference to “a capture agent” includes a singlecapture agent and multiple capture agents, reference to “a detectionagent” includes a single detection agent and multiple detection agents,and reference to “an agent” includes a single agent and multiple agents.

As used herein, the terms “adapted” and “configured” mean that theelement, component, or other subject matter is designed and/or intendedto perform a given function. Thus, the use of the terms “adapted” and“configured” should not be construed to mean that a given element,component, or other subject matter is simply “capable of” performing agiven function. Similarly, subject matter that is recited as beingconfigured to perform a particular function may additionally oralternatively be described as being operative to perform that function.

As used herein, the phrase, “for example,” the phrase, “as an example,”and/or simply the terms “example” and “exemplary” when used withreference to one or more components, features, details, structures,embodiments, and/or methods according to the present disclosure, areintended to convey that the described component, feature, detail,structure, embodiment, and/or method is an illustrative, non-exclusiveexample of components, features, details, structures, embodiments,and/or methods according to the present disclosure. Thus, the describedcomponent, feature, detail, structure, embodiment, and/or method is notintended to be limiting, required, or exclusive/exhaustive; and othercomponents, features, details, structures, embodiments, and/or methods,including structurally and/or functionally similar and/or equivalentcomponents, features, details, structures, embodiments, and/or methods,are also within the scope of the present disclosure.

As used herein, the phrases “at least one of” and “one or more of,” inreference to a list of more than one entity, means any one or more ofthe entity in the list of entity, and is not limited to at least one ofeach and every entity specifically listed within the list of entity. Forexample, “at least one of A and B” (or, equivalently, “at least one of Aor B,” or, equivalently, “at least one of A and/or B”) may refer to Aalone, B alone, or the combination of A and B.

As used herein, the term “and/or” placed between a first entity and asecond entity means one of (1) the first entity, (2) the second entity,and (3) the first entity and the second entity. Multiple entity listedwith “and/or” should be construed in the same manner, i.e., “one ormore” of the entity so conjoined. Other entity may optionally be presentother than the entity specifically identified by the “and/or” clause,whether related or unrelated to those entities specifically identified.

Where numerical ranges are mentioned herein, the invention includesembodiments in which the endpoints are included, embodiments in whichboth endpoints are excluded, and embodiments in which one endpoint isincluded and the other is excluded. It should be assumed that bothendpoints are included unless indicated otherwise. Furthermore, unlessotherwise indicated or otherwise evident from the context andunderstanding of one of ordinary skill in the art.

In the event that any patents, patent applications, or other referencesare incorporated by reference herein and (1) define a term in a mannerthat is inconsistent with and/or (2) are otherwise inconsistent with,either the non-incorporated portion of the present disclosure or any ofthe other incorporated references, the non-incorporated portion of thepresent disclosure shall control, and the term or incorporateddisclosure therein shall only control with respect to the reference inwhich the term is defined and/or the incorporated disclosure was presentoriginally.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims.

What is claimed is:
 1. An apparatus for assaying an analyte in adeformable sample, comprising: (a) a sample holder comprising twoplates, wherein the two plates sandwich the sample between the samplecontact areas of the plates in a thin layer, (b) a plurality ofreference marks on the sample contact areas of the plates, wherein atleast one of the geometric properties and/or the optical properties ofthe reference marks is predetermined and known; (c) an imager thatimages the sample contact area; (d) a non-transient computer medium thatcomprises a machine learning algorithm that uses the images withreference marks.
 2. A method of assaying an analyte in a deformablesample, comprising: (a) obtaining the apparatus of claim 1; (b) placingthe sample into the sample holder, wherein at least a part of the sampleis between the two plates; (c) taking, using the imager, one or moreimages of the sample and the reference markers; and (d) detecting theanalyte by analyzing the one or more images and the machine learningalgorithm.
 3. The method of claim 2, wherein the one or more imagesconsist of both bright images and fluorescent images.
 4. The apparatusof claim 1, wherein the reference marks comprise the spacers, whereinthe spacers are between the two plates and regulate the gap between thetwo plates; and wherein the spacers are pillars.
 5. The apparatus ofclaim 1, wherein the reference marks are periodic and the period ispredetermined and known.
 6. The method of claim 2, wherein the analyzingof each image comprises determining a rotational orientation of one ormore of the reference marks about the axis relative to the imagingsystem.
 7. The method of claim 2, wherein the analyzing of each imagecomprises comparing image information about the reference marks to aprior knowledge about the reference marks.
 8. The apparatus of claim 1,wherein the sample contact area further comprises the detection agentcomprising antibodies configured to specifically bind to protein analytein the sample.
 9. The apparatus of claim 1, wherein the sample contactarea further comprises the detection agent comprising oligonucleotideprobes configured to specifically bind to DNA and/or RNA in the sample.10. The apparatus of claim 1, wherein one or both plates is flexible,and wherein for the fourth power of the inter-spacer-distance (ISD)divided by the thickness of the flexible plate (h) and the Young'smodulus (E) of the flexible plate, ISD⁴/(hE), is less than 10⁵ um³/GPa,less than 10⁴ um³/GPa or less than 10³ um³/GPa.
 11. The apparatus ofclaim 1, wherein the sample is selected from the group consisting of:amniotic fluid, aqueous humour, vitreous humour, blood (e.g., wholeblood, fractionated blood, plasma or serum), breast milk, cerebrospinalfluid (CSF), cerumen (earwax), chyle, chime, endolymph, perilymph,feces, breath, gastric acid, gastric juice, lymph, mucus (includingnasal drainage and phlegm), pericardial fluid, peritoneal fluid, pleuralfluid, pus, rheum, saliva, exhaled breath condensates, sebum, semen,sputum, sweat, synovial fluid, tears, vomit, urine, and any combinationthereof.
 12. The method of claim 2, wherein the reference marks comprisethe spacers, wherein the spacers are between the two plates and regulatethe gap between the two plates; and wherein the spacers are pillars. 13.The method of claim 2, wherein the reference marks are periodic and theperiod is predetermined and known.
 14. The method of claim 2, whereinthe sample contact area further comprises the detection agent comprisingantibodies configured to specifically bind to protein analyte in thesample.
 15. The method of claim 2, wherein the sample contact areafurther comprises the detection agent comprising oligonucleotide probesconfigured to specifically bind to DNA and/or RNA in the sample.
 16. Themethod of claim 2, wherein one or both plates is flexible, and whereinfor the fourth power of the inter-spacer-distance (ISD) divided by thethickness of the flexible plate (h) and the Young's modulus (E) of theflexible plate, ISD⁴/(hE), is less than 10⁵ um³/GPa, less than 10⁴um³/GPa or less than 10³ um³/GPa.
 17. The method of claim 2, wherein thesample is selected from the group consisting of: amniotic fluid, aqueoushumour, vitreous humour, blood (e.g., whole blood, fractionated blood,plasma or serum), breast milk, cerebrospinal fluid (CSF), cerumen(earwax), chyle, chime, endolymph, perilymph, feces, breath, gastricacid, gastric juice, lymph, mucus (including nasal drainage and phlegm),pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, saliva,exhaled breath condensates, sebum, semen, sputum, sweat, synovial fluid,tears, vomit, urine, and any combination thereof.